DEFYYOURDNA

HowtheNewGenePatchPersonalizedMedicines

WillHelpYouOvercomemYourGreatestHealthChallenges

STEPHENBSHREWSBURYMD

10FingerPressElCerritoCA

copy2013StephenBShrewsbury

AllrightsreservedNopartofthisbookmaybereproducedortransmittedinanyformorbyanymeanselectronicormechanicalincludingphotocopyingrecordingorbyanyinformationstorageandretrievalsystemexceptinthecaseofbriefquotationsembodiedincriticalarticlesandreviewswithoutpriorwrittenpermissionofthepublisherAlthoughtheauthorandpublisherhavemadeeveryefforttoensuretheaccuracyandcompletenessofinformationcontainedinthisbookweassumenoresponsibilityforerrorsinaccuraciesomissionsoranyinconsistencyherein

wwwdefyyourdnabookcomwwwfacebookcomdefyyourdnabook

PrintedintheUnitedStatesofAmerica

Publishedby10FingerPressDistributedtothebooktradebyMidpointTradeBooksEditedbyTheAuthorsTeam(wwwAuthorsTeamcom)InteriorDesignLayoutGhislainViauISBN9781933174860

LibraryofCongressControlNumber2013903576

PublisherrsquosCataloging-in-Publication(ProvidedbyQualityBooksInc)

ShrewsburyStephenBDefyyourDNAhowthenewgenepatchpersonalizedmedicineswillhelpyouovercomeyourgreatesthealthchallengesStephenBShrewsburypcmIncludesbibliographicalreferencesandindex

1Antisensenucleicacids--Therapeuticuse--Popularworks2Oligonucleotides--Therapeuticuse--Popularworks3Recombinantmolecules--Therapeuticuse--Popularworks4Pharmaceuticalbiotechnology--PopularworksITitle

RM666A564S572013 6153rsquo1QBI13-200003

090807060504 54321

copy2013StephenBShrewsbury

AllrightsreservedNopartofthisbookmaybereproducedortransmittedinanyformorbyanymeanselectronicormechanicalincludingphotocopyingrecordingorbyanyinformationstorageandretrievalsystemexceptinthecaseofbriefquotationsembodiedincriticalarticlesandreviewswithoutpriorwrittenpermissionofthepublisherAlthoughtheauthorandpublisherhavemadeeveryefforttoensuretheaccuracyandcompletenessofinformationcontainedinthisbookweassumenoresponsibilityforerrorsinaccuraciesomissionsoranyinconsistencyherein

wwwdefyyourdnabookcomwwwfacebookcomdefyyourdnabook

PrintedintheUnitedStatesofAmerica

Publishedby10FingerPressDistributedtothebooktradebyMidpointTradeBooksEditedbyTheAuthorsTeam(wwwAuthorsTeamcom)InteriorDesignLayoutGhislainViauISBN9781933174860

LibraryofCongressControlNumber2013903576

PublisherrsquosCataloging-in-Publication(ProvidedbyQualityBooksInc)

ShrewsburyStephenBDefyyourDNAhowthenewgenepatchpersonalizedmedicineswillhelpyouovercomeyourgreatesthealthchallengesStephenBShrewsburypcmIncludesbibliographicalreferencesandindex

1Antisensenucleicacids--Therapeuticuse--Popularworks2Oligonucleotides--Therapeuticuse--Popularworks3Recombinantmolecules--Therapeuticuse--Popularworks4Pharmaceuticalbiotechnology--PopularworksITitle

RM666A564S572013 6153rsquo1QBI13-200003

090807060504 54321

TableofContents

Acknowledgements

Preface

OneMedicineOntheBrink

TwoMedicinethroughtheMillennia

ThreeFromPrimordialSouptoPersonalizedMedicine

FourCamouflageYourGenes

FiveTheEndofHereditaryRareDisease

SixNewDrugsforBadBugs

SevenTheBigOnesCommonChronicDiseases

EightPhysiciansPastPresentandFuture

NineHowAreNewDrugsRegulated

TenThePharmacyoftheFuture

ElevenToInfinityandBeyond

Appendices

GlossaryofTerms

BibliographyReferencesandsuggestedreading

Index

ThisbookisdedicatedtothemanyhealthyvolunteerspatientsandtheirfamilieswhohaveparticipatedinanydrugstudyWithoutyoutherewouldbenonewdrugsWithoutyoutherewouldbe

noadvancesinpharmaceuticalsYouaretheoneswhoareselflesslybringinghopetothemillionsofpatientsaroundtheworldwaitingfortomorrowrsquosadvancesintherapeutics

Acknowledgements

IamindebtedtoProfessor DwightWeller who as Senior Vice President has headed the

chemistry department at AVI BioPharma (as it was) since leaving OregonState University He has taught me all I know about the chemistry ofoligomersandthefactthatthatisnotmuchisnodiscredittohimHisreviewofthismanuscripthoweverhasbeenasusualmeticulousandconstructive

Professor Ryszard Kole who worked alongside the Nobel LaureateProfessorSidAltmanthroughthediscoveryofldquoalternativesplicingrdquoatYaleUniversityRyszardwasSeniorVicePresidentofResearchandDistinguishedScientist during my time at AVI BioPharma and kindly reviewed andprovided input to this manuscript as well as teachingmemore about exonskippingthanIeverexpectedtograsp

DrPatrickIversenwasSeniorVicePresidentofResearchandInnovationwhenIwasatAVIBioPharmaandmysquashpartner inCorvallisPathasbeenthetirelessdrivingforcebehindtheanti-microbialresearchfortheAVIstable of oligomers and found time to review and comment on the relevantsectionsofthisbook

MaheshGrossmanandhiscolleaguesatTheAuthorsTeamhavehelpedmemakethebookmorereadableandreducesentencelengthcomplexityanduseof toomuch jargonHis supportas thechaptershaveebbedand flowedandeachhasbeenscrubbedandbuffedoverthelastsixteenmonthshasbeenwelcome

Merry Shiyu Wang has provided excellent illustrations and has beenapproachable expeditious and efficient turning out diagrams to my designwhilemakingthemscientificallyaccurateandpleasingtotheeyeThankyou

S

Preface

ome years ago I was a family doctor in Evesham a quaint medievalmarket town on the banks of the River Avon in Worcestershire in the

UnitedKingdom Iwasgetting to the endofmymorning surgerywhen thephonerangAworriedpatientwashopingIcouldseeherson

IknewthepatientMrsSinghandhersonDeepakasIdidmostofmypatientsButlikemostparentsshedidnrsquotcomeveryoftensoIdidnotknowherwellHersonwasjustoverthreeandIhadlookedafterMrsSinghandherhusbandsincebeforeshegavebirthtoherdaughterJasminderfiveyearsearlierDeepakhadbeenahappyhealthybabywhohadpassedhis18-monthassessmentwithmedespitebeingalittleslowinwalking

Mrs Singh came in pushing a grinning and content three-year-old in astrollerAsDeepakstaredatmeIwonderedifmaybehewasjustenjoyingalittleextraattentionMrsSinghdescribedhowhersonhadbecomereluctanttowalkintheafternoonsandeveningsShewasworriedthathehadhurthislegorperhapshadaninfection

IturnedtoDeepaksquattingdowninfrontofhisstrollersothatmyeyeswereonhislevelldquoWhathaveyougotthererdquoIaskedpointingtoatattytoyhe was holding which I suspected had once resembled a tiger Deepakbeamedbutsaidnothing

HopingtoenticehimtowalkIwalkedovertoalargetreasurechestofmyownchildrenrsquosdiscardedtoysthatwerestowedbeneathacouchIpulledoutasqueakingelephanttoyThatgothisattention

ldquoLookDeepakrdquoIofferedpokingtheheadoftheelephantroundthelidof the box and then quickly withdrawing it and squeezing it forcefullyDeepak immediatelydroppedhis tigerand raisedhimselfoutofhis strollerHewaddledacrossmyexaminationroomtowardme-onlyafewstepsbutheseemed indeed rather reluctant My heart sank This was not the nimbleconfidentstrideofanormalhealthythree-year-old

Igavehimtheelephantandhesatdownwithathump

Iwent back tomydesk andquickly scannedDeepakrsquosmedical recordsThere was nothing to suggest he was not entirely normal I quizzed MrsSinghaboutthehealthofherimmediatefamilybutagaintherewasnothingnotable I continued eyeing Deepakwhowas playing contentedly Then herolledoverandstruggledtohisfeet

WiththatIwasalarmedAt medical school students are taught about rare diseases but most

doctors especially those with a list of less than 3000 patients never see asingle case ofmost of themSo thememory fadesBut couldDeepak haveDuchennemuscular dystrophy It is very rare It onlyoccurs in boys oftenwith no family history and is often undetectable at birth It could presentprettymuchthewayDeepakappearednowwithdifficultywalkingataroundagethreewithwhatlookedlikeenlargedcalfmusclesonhislegs

I got down on my hands and knees and looked carefully at the nowstandingDeepakwhohadbecomeinterestedinacolorfulpaperweightonmydeskCertainlyhehad largecalfmuscles fora three-year-oldQuitechunkylittlelegsbutweretheyabnormallyso

Thiswould be a complex diagnosis tomake and a terrible blow to theSinghs The disease gradually robs boys of muscle power They lose theabilitytowalkandlatertoevenstandwithbracesbecomingdependentonawheelchairformobilityThentheygetbreathingtroubleandrequireportableventilatorsBut despite all the support they become totally unable tomoveandoftendieintheirearlytwentieswhentheirheartmusclealsofailsiftheysurvivethatlong

Little progress has been made on this terrible disease since it was firstdescribedby theFrenchneurologistGuillaumeDuchenne in1891DespiteintensestudybyscientistsanddoctorsoverthelastcenturythereisnocureIn1986scientistsdiscoveredanyoneofmanymutationsinasinglegenecancausethemusclecellsoftheseboystofailtomakeavitalproteinAsaresulttheir muscles waste away High doses of steroids temporarily delay theinevitablebutnoeffectivetreatmentexistedthenornowandsteroidsbringproblemsoftheirown

ItisaterribleunforgivingdiseaseandtheknowledgethatthebestIcoulddo if I was correct was to watch Deepak slip away is one of the mostunpleasantprofessionalsituationsadoctorcanencounter

Therapeutic impotence is something we were taught to cope with at

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

ThisbookisdedicatedtothemanyhealthyvolunteerspatientsandtheirfamilieswhohaveparticipatedinanydrugstudyWithoutyoutherewouldbenonewdrugsWithoutyoutherewouldbe

noadvancesinpharmaceuticalsYouaretheoneswhoareselflesslybringinghopetothemillionsofpatientsaroundtheworldwaitingfortomorrowrsquosadvancesintherapeutics

Acknowledgements

IamindebtedtoProfessor DwightWeller who as Senior Vice President has headed the

chemistry department at AVI BioPharma (as it was) since leaving OregonState University He has taught me all I know about the chemistry ofoligomersandthefactthatthatisnotmuchisnodiscredittohimHisreviewofthismanuscripthoweverhasbeenasusualmeticulousandconstructive

Professor Ryszard Kole who worked alongside the Nobel LaureateProfessorSidAltmanthroughthediscoveryofldquoalternativesplicingrdquoatYaleUniversityRyszardwasSeniorVicePresidentofResearchandDistinguishedScientist during my time at AVI BioPharma and kindly reviewed andprovided input to this manuscript as well as teachingmemore about exonskippingthanIeverexpectedtograsp

DrPatrickIversenwasSeniorVicePresidentofResearchandInnovationwhenIwasatAVIBioPharmaandmysquashpartner inCorvallisPathasbeenthetirelessdrivingforcebehindtheanti-microbialresearchfortheAVIstable of oligomers and found time to review and comment on the relevantsectionsofthisbook

MaheshGrossmanandhiscolleaguesatTheAuthorsTeamhavehelpedmemakethebookmorereadableandreducesentencelengthcomplexityanduseof toomuch jargonHis supportas thechaptershaveebbedand flowedandeachhasbeenscrubbedandbuffedoverthelastsixteenmonthshasbeenwelcome

Merry Shiyu Wang has provided excellent illustrations and has beenapproachable expeditious and efficient turning out diagrams to my designwhilemakingthemscientificallyaccurateandpleasingtotheeyeThankyou

S

Preface

ome years ago I was a family doctor in Evesham a quaint medievalmarket town on the banks of the River Avon in Worcestershire in the

UnitedKingdom Iwasgetting to the endofmymorning surgerywhen thephonerangAworriedpatientwashopingIcouldseeherson

IknewthepatientMrsSinghandhersonDeepakasIdidmostofmypatientsButlikemostparentsshedidnrsquotcomeveryoftensoIdidnotknowherwellHersonwasjustoverthreeandIhadlookedafterMrsSinghandherhusbandsincebeforeshegavebirthtoherdaughterJasminderfiveyearsearlierDeepakhadbeenahappyhealthybabywhohadpassedhis18-monthassessmentwithmedespitebeingalittleslowinwalking

Mrs Singh came in pushing a grinning and content three-year-old in astrollerAsDeepakstaredatmeIwonderedifmaybehewasjustenjoyingalittleextraattentionMrsSinghdescribedhowhersonhadbecomereluctanttowalkintheafternoonsandeveningsShewasworriedthathehadhurthislegorperhapshadaninfection

IturnedtoDeepaksquattingdowninfrontofhisstrollersothatmyeyeswereonhislevelldquoWhathaveyougotthererdquoIaskedpointingtoatattytoyhe was holding which I suspected had once resembled a tiger Deepakbeamedbutsaidnothing

HopingtoenticehimtowalkIwalkedovertoalargetreasurechestofmyownchildrenrsquosdiscardedtoysthatwerestowedbeneathacouchIpulledoutasqueakingelephanttoyThatgothisattention

ldquoLookDeepakrdquoIofferedpokingtheheadoftheelephantroundthelidof the box and then quickly withdrawing it and squeezing it forcefullyDeepak immediatelydroppedhis tigerand raisedhimselfoutofhis strollerHewaddledacrossmyexaminationroomtowardme-onlyafewstepsbutheseemed indeed rather reluctant My heart sank This was not the nimbleconfidentstrideofanormalhealthythree-year-old

Igavehimtheelephantandhesatdownwithathump

Iwent back tomydesk andquickly scannedDeepakrsquosmedical recordsThere was nothing to suggest he was not entirely normal I quizzed MrsSinghaboutthehealthofherimmediatefamilybutagaintherewasnothingnotable I continued eyeing Deepakwhowas playing contentedly Then herolledoverandstruggledtohisfeet

WiththatIwasalarmedAt medical school students are taught about rare diseases but most

doctors especially those with a list of less than 3000 patients never see asingle case ofmost of themSo thememory fadesBut couldDeepak haveDuchennemuscular dystrophy It is very rare It onlyoccurs in boys oftenwith no family history and is often undetectable at birth It could presentprettymuchthewayDeepakappearednowwithdifficultywalkingataroundagethreewithwhatlookedlikeenlargedcalfmusclesonhislegs

I got down on my hands and knees and looked carefully at the nowstandingDeepakwhohadbecomeinterestedinacolorfulpaperweightonmydeskCertainlyhehad largecalfmuscles fora three-year-oldQuitechunkylittlelegsbutweretheyabnormallyso

Thiswould be a complex diagnosis tomake and a terrible blow to theSinghs The disease gradually robs boys of muscle power They lose theabilitytowalkandlatertoevenstandwithbracesbecomingdependentonawheelchairformobilityThentheygetbreathingtroubleandrequireportableventilatorsBut despite all the support they become totally unable tomoveandoftendieintheirearlytwentieswhentheirheartmusclealsofailsiftheysurvivethatlong

Little progress has been made on this terrible disease since it was firstdescribedby theFrenchneurologistGuillaumeDuchenne in1891DespiteintensestudybyscientistsanddoctorsoverthelastcenturythereisnocureIn1986scientistsdiscoveredanyoneofmanymutationsinasinglegenecancausethemusclecellsoftheseboystofailtomakeavitalproteinAsaresulttheir muscles waste away High doses of steroids temporarily delay theinevitablebutnoeffectivetreatmentexistedthenornowandsteroidsbringproblemsoftheirown

ItisaterribleunforgivingdiseaseandtheknowledgethatthebestIcoulddo if I was correct was to watch Deepak slip away is one of the mostunpleasantprofessionalsituationsadoctorcanencounter

Therapeutic impotence is something we were taught to cope with at

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

Acknowledgements

IamindebtedtoProfessor DwightWeller who as Senior Vice President has headed the

chemistry department at AVI BioPharma (as it was) since leaving OregonState University He has taught me all I know about the chemistry ofoligomersandthefactthatthatisnotmuchisnodiscredittohimHisreviewofthismanuscripthoweverhasbeenasusualmeticulousandconstructive

Professor Ryszard Kole who worked alongside the Nobel LaureateProfessorSidAltmanthroughthediscoveryofldquoalternativesplicingrdquoatYaleUniversityRyszardwasSeniorVicePresidentofResearchandDistinguishedScientist during my time at AVI BioPharma and kindly reviewed andprovided input to this manuscript as well as teachingmemore about exonskippingthanIeverexpectedtograsp

DrPatrickIversenwasSeniorVicePresidentofResearchandInnovationwhenIwasatAVIBioPharmaandmysquashpartner inCorvallisPathasbeenthetirelessdrivingforcebehindtheanti-microbialresearchfortheAVIstable of oligomers and found time to review and comment on the relevantsectionsofthisbook

MaheshGrossmanandhiscolleaguesatTheAuthorsTeamhavehelpedmemakethebookmorereadableandreducesentencelengthcomplexityanduseof toomuch jargonHis supportas thechaptershaveebbedand flowedandeachhasbeenscrubbedandbuffedoverthelastsixteenmonthshasbeenwelcome

Merry Shiyu Wang has provided excellent illustrations and has beenapproachable expeditious and efficient turning out diagrams to my designwhilemakingthemscientificallyaccurateandpleasingtotheeyeThankyou

S

Preface

ome years ago I was a family doctor in Evesham a quaint medievalmarket town on the banks of the River Avon in Worcestershire in the

UnitedKingdom Iwasgetting to the endofmymorning surgerywhen thephonerangAworriedpatientwashopingIcouldseeherson

IknewthepatientMrsSinghandhersonDeepakasIdidmostofmypatientsButlikemostparentsshedidnrsquotcomeveryoftensoIdidnotknowherwellHersonwasjustoverthreeandIhadlookedafterMrsSinghandherhusbandsincebeforeshegavebirthtoherdaughterJasminderfiveyearsearlierDeepakhadbeenahappyhealthybabywhohadpassedhis18-monthassessmentwithmedespitebeingalittleslowinwalking

Mrs Singh came in pushing a grinning and content three-year-old in astrollerAsDeepakstaredatmeIwonderedifmaybehewasjustenjoyingalittleextraattentionMrsSinghdescribedhowhersonhadbecomereluctanttowalkintheafternoonsandeveningsShewasworriedthathehadhurthislegorperhapshadaninfection

IturnedtoDeepaksquattingdowninfrontofhisstrollersothatmyeyeswereonhislevelldquoWhathaveyougotthererdquoIaskedpointingtoatattytoyhe was holding which I suspected had once resembled a tiger Deepakbeamedbutsaidnothing

HopingtoenticehimtowalkIwalkedovertoalargetreasurechestofmyownchildrenrsquosdiscardedtoysthatwerestowedbeneathacouchIpulledoutasqueakingelephanttoyThatgothisattention

ldquoLookDeepakrdquoIofferedpokingtheheadoftheelephantroundthelidof the box and then quickly withdrawing it and squeezing it forcefullyDeepak immediatelydroppedhis tigerand raisedhimselfoutofhis strollerHewaddledacrossmyexaminationroomtowardme-onlyafewstepsbutheseemed indeed rather reluctant My heart sank This was not the nimbleconfidentstrideofanormalhealthythree-year-old

Igavehimtheelephantandhesatdownwithathump

Iwent back tomydesk andquickly scannedDeepakrsquosmedical recordsThere was nothing to suggest he was not entirely normal I quizzed MrsSinghaboutthehealthofherimmediatefamilybutagaintherewasnothingnotable I continued eyeing Deepakwhowas playing contentedly Then herolledoverandstruggledtohisfeet

WiththatIwasalarmedAt medical school students are taught about rare diseases but most

doctors especially those with a list of less than 3000 patients never see asingle case ofmost of themSo thememory fadesBut couldDeepak haveDuchennemuscular dystrophy It is very rare It onlyoccurs in boys oftenwith no family history and is often undetectable at birth It could presentprettymuchthewayDeepakappearednowwithdifficultywalkingataroundagethreewithwhatlookedlikeenlargedcalfmusclesonhislegs

I got down on my hands and knees and looked carefully at the nowstandingDeepakwhohadbecomeinterestedinacolorfulpaperweightonmydeskCertainlyhehad largecalfmuscles fora three-year-oldQuitechunkylittlelegsbutweretheyabnormallyso

Thiswould be a complex diagnosis tomake and a terrible blow to theSinghs The disease gradually robs boys of muscle power They lose theabilitytowalkandlatertoevenstandwithbracesbecomingdependentonawheelchairformobilityThentheygetbreathingtroubleandrequireportableventilatorsBut despite all the support they become totally unable tomoveandoftendieintheirearlytwentieswhentheirheartmusclealsofailsiftheysurvivethatlong

Little progress has been made on this terrible disease since it was firstdescribedby theFrenchneurologistGuillaumeDuchenne in1891DespiteintensestudybyscientistsanddoctorsoverthelastcenturythereisnocureIn1986scientistsdiscoveredanyoneofmanymutationsinasinglegenecancausethemusclecellsoftheseboystofailtomakeavitalproteinAsaresulttheir muscles waste away High doses of steroids temporarily delay theinevitablebutnoeffectivetreatmentexistedthenornowandsteroidsbringproblemsoftheirown

ItisaterribleunforgivingdiseaseandtheknowledgethatthebestIcoulddo if I was correct was to watch Deepak slip away is one of the mostunpleasantprofessionalsituationsadoctorcanencounter

Therapeutic impotence is something we were taught to cope with at

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

S

Preface

ome years ago I was a family doctor in Evesham a quaint medievalmarket town on the banks of the River Avon in Worcestershire in the

UnitedKingdom Iwasgetting to the endofmymorning surgerywhen thephonerangAworriedpatientwashopingIcouldseeherson

IknewthepatientMrsSinghandhersonDeepakasIdidmostofmypatientsButlikemostparentsshedidnrsquotcomeveryoftensoIdidnotknowherwellHersonwasjustoverthreeandIhadlookedafterMrsSinghandherhusbandsincebeforeshegavebirthtoherdaughterJasminderfiveyearsearlierDeepakhadbeenahappyhealthybabywhohadpassedhis18-monthassessmentwithmedespitebeingalittleslowinwalking

Mrs Singh came in pushing a grinning and content three-year-old in astrollerAsDeepakstaredatmeIwonderedifmaybehewasjustenjoyingalittleextraattentionMrsSinghdescribedhowhersonhadbecomereluctanttowalkintheafternoonsandeveningsShewasworriedthathehadhurthislegorperhapshadaninfection

IturnedtoDeepaksquattingdowninfrontofhisstrollersothatmyeyeswereonhislevelldquoWhathaveyougotthererdquoIaskedpointingtoatattytoyhe was holding which I suspected had once resembled a tiger Deepakbeamedbutsaidnothing

HopingtoenticehimtowalkIwalkedovertoalargetreasurechestofmyownchildrenrsquosdiscardedtoysthatwerestowedbeneathacouchIpulledoutasqueakingelephanttoyThatgothisattention

ldquoLookDeepakrdquoIofferedpokingtheheadoftheelephantroundthelidof the box and then quickly withdrawing it and squeezing it forcefullyDeepak immediatelydroppedhis tigerand raisedhimselfoutofhis strollerHewaddledacrossmyexaminationroomtowardme-onlyafewstepsbutheseemed indeed rather reluctant My heart sank This was not the nimbleconfidentstrideofanormalhealthythree-year-old

Igavehimtheelephantandhesatdownwithathump

Iwent back tomydesk andquickly scannedDeepakrsquosmedical recordsThere was nothing to suggest he was not entirely normal I quizzed MrsSinghaboutthehealthofherimmediatefamilybutagaintherewasnothingnotable I continued eyeing Deepakwhowas playing contentedly Then herolledoverandstruggledtohisfeet

WiththatIwasalarmedAt medical school students are taught about rare diseases but most

doctors especially those with a list of less than 3000 patients never see asingle case ofmost of themSo thememory fadesBut couldDeepak haveDuchennemuscular dystrophy It is very rare It onlyoccurs in boys oftenwith no family history and is often undetectable at birth It could presentprettymuchthewayDeepakappearednowwithdifficultywalkingataroundagethreewithwhatlookedlikeenlargedcalfmusclesonhislegs

I got down on my hands and knees and looked carefully at the nowstandingDeepakwhohadbecomeinterestedinacolorfulpaperweightonmydeskCertainlyhehad largecalfmuscles fora three-year-oldQuitechunkylittlelegsbutweretheyabnormallyso

Thiswould be a complex diagnosis tomake and a terrible blow to theSinghs The disease gradually robs boys of muscle power They lose theabilitytowalkandlatertoevenstandwithbracesbecomingdependentonawheelchairformobilityThentheygetbreathingtroubleandrequireportableventilatorsBut despite all the support they become totally unable tomoveandoftendieintheirearlytwentieswhentheirheartmusclealsofailsiftheysurvivethatlong

Little progress has been made on this terrible disease since it was firstdescribedby theFrenchneurologistGuillaumeDuchenne in1891DespiteintensestudybyscientistsanddoctorsoverthelastcenturythereisnocureIn1986scientistsdiscoveredanyoneofmanymutationsinasinglegenecancausethemusclecellsoftheseboystofailtomakeavitalproteinAsaresulttheir muscles waste away High doses of steroids temporarily delay theinevitablebutnoeffectivetreatmentexistedthenornowandsteroidsbringproblemsoftheirown

ItisaterribleunforgivingdiseaseandtheknowledgethatthebestIcoulddo if I was correct was to watch Deepak slip away is one of the mostunpleasantprofessionalsituationsadoctorcanencounter

Therapeutic impotence is something we were taught to cope with at

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

Iwent back tomydesk andquickly scannedDeepakrsquosmedical recordsThere was nothing to suggest he was not entirely normal I quizzed MrsSinghaboutthehealthofherimmediatefamilybutagaintherewasnothingnotable I continued eyeing Deepakwhowas playing contentedly Then herolledoverandstruggledtohisfeet

WiththatIwasalarmedAt medical school students are taught about rare diseases but most

doctors especially those with a list of less than 3000 patients never see asingle case ofmost of themSo thememory fadesBut couldDeepak haveDuchennemuscular dystrophy It is very rare It onlyoccurs in boys oftenwith no family history and is often undetectable at birth It could presentprettymuchthewayDeepakappearednowwithdifficultywalkingataroundagethreewithwhatlookedlikeenlargedcalfmusclesonhislegs

I got down on my hands and knees and looked carefully at the nowstandingDeepakwhohadbecomeinterestedinacolorfulpaperweightonmydeskCertainlyhehad largecalfmuscles fora three-year-oldQuitechunkylittlelegsbutweretheyabnormallyso

Thiswould be a complex diagnosis tomake and a terrible blow to theSinghs The disease gradually robs boys of muscle power They lose theabilitytowalkandlatertoevenstandwithbracesbecomingdependentonawheelchairformobilityThentheygetbreathingtroubleandrequireportableventilatorsBut despite all the support they become totally unable tomoveandoftendieintheirearlytwentieswhentheirheartmusclealsofailsiftheysurvivethatlong

Little progress has been made on this terrible disease since it was firstdescribedby theFrenchneurologistGuillaumeDuchenne in1891DespiteintensestudybyscientistsanddoctorsoverthelastcenturythereisnocureIn1986scientistsdiscoveredanyoneofmanymutationsinasinglegenecancausethemusclecellsoftheseboystofailtomakeavitalproteinAsaresulttheir muscles waste away High doses of steroids temporarily delay theinevitablebutnoeffectivetreatmentexistedthenornowandsteroidsbringproblemsoftheirown

ItisaterribleunforgivingdiseaseandtheknowledgethatthebestIcoulddo if I was correct was to watch Deepak slip away is one of the mostunpleasantprofessionalsituationsadoctorcanencounter

Therapeutic impotence is something we were taught to cope with at

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

medical school However lectures canrsquot prepare a doctor for the horror oftelling a mother the devastating future she and her family may face Howcould I explain that we could put men on the moon but I could not stopDeepakfromslowlydyingoverthenexttwentyyearsaseverymuscleinhisbodystoppedworking

ManyyearslaterthathorriblefeelingremainsfreshAt the time I thought of my own son who had been born with a rare

diseasemulticysticdysplastickidneyHewasexpected todiesoonafterhisbirthandmyworldhadchangedwithhisarrival

He survived and thrives to this day which is not how it would be forDeepakifhehadDuchennemusculardystrophy

I continued to chat withMrs Singh while I desperately considered theother possible diseases illnesses and injuries that could have befallenDeepakNone of them seemed likelyAttempting nonchalance I convincedMrsSinghoftheneedforabloodtestanddrewthesamplefromastrugglingandhowlingDeepak

Severalweeks later theworstcasescenariowasconfirmedDeepakhadDuchenne muscular dystrophy (DMD) The diagnosis was understandablydifficult for theSinghs to absorbNoparentwants to limit the dreams theyhavefor theirchildrenmuch lessaccept that theywould likelyoutlive theiroffspring

Later that same evening after reading stories to my own children andmarveling at my own son who had miraculously survived the fate that hisdoctors had predicted for him I wondered if we would ever develop atreatmentletaloneacureforDMDByweImeantallthescientistsdoctorslaboratoryworkersandeveryoneelseinvolvedinhealthcare

Deepak may be dead now or struggling in a wheelchair and on aventilator What more could I have done for him back then NothingAbsolutelynothingIcouldnrsquotevenofferhopeThatwasperhapsthehardestlessonandmostunpleasantdutyIeverhadasadoctor

Howevernowin2012IamconfidentthatphysicianswillsoonbeabletooffermorethanhopetofamiliesliketheSinghrsquosMaybenotthisyearandmaybenotnextbutsoonThehopecomesfromanewclassofmedicinesthatcan patch the defective message that comes from a faulty gene that causediseases like Duchennersquos Some of these patches work by stopping theproductionofadiseasecausingproteineitherwithinthesickpatientorbyan

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

invadingbacteriaorvirusOthersofthesenewmedicinesworkbytriggeringanaturally occurring alternative process in the human cell to produce a newversion of a vital missing protein This book focuses mostly on this lattercategory which offers great hope In the future these new gene patchmedicineswillallowyoutodefyyourDNA

Myeldestsonqualifiedasadoctorhimselfin2011BecauseofthesenewmedicineshewillbeabletoofferhopetoALLofhispatientsnomatterwhattheirdiagnosis In thenot toodistantfuturewhenachild isborn theyrsquollberequiredtohavetwodocumentsabirthcertificateandamapoftheirDNAThemapwillshowwhichroadstotaketogettotheirdestinationandwhichtoavoid Avoiding a bad ending may involve behavioral modification ortraditionalmedicinesorthehelpofoneofthesenewgene-patcholigomersasthey gain acceptance They are like molecular Band Aids that will hidedamagedpiecesofthegeneticmessage

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare

T

ChapterThree

FromPrimordialSouptoPersonalizedMedicine

he building blocks of life on earth began in the hot reactive stew ofhydrogen carbon oxygen and nitrogen that defined our planet roughly

threeandahalfbillionyearsagoThatwasameretenbillionyearsaftertheuniversewasformedbytheBigBangandonebillionyearsafterourplanethad formed It is there that the rules governing genetics and personalizedmedicinespawned

YetitwasonlyduringthelastonehundredyearsthatscientistsconcludedthatDNAwasthesourceofgeneticinheritance

GenesandDNAThinkofgenesaslibrarybooksstackedonshelves(thechromosomes)in

a library (the nucleus) of a city (the cell)All free livingorganisms containgenes frombacteria to the simplest algaeandplants rightup thevegetableandanimalkingdoms toushomo sapiens at the self-proclaimed topof theevolutionary tree Even viruses which are not really alive and can onlyreproduceandmanufacturetheirproteinsinsidethehostcellrequireatleastafewgenescomposedofeitherDNAorRNA

SowhatexactlyisDNAdeoxyribonucleicacidItrsquosapolymerwhichisalongchainofconnectedunitsliketheteethofazipperorastringofbeadsonanecklaceDNAwasdiscoveredbyFriedrichMiescheraSwissphysicianinthe1860sHeobtainedcellsfromthepusfromdiscardedsurgicalbandagesand isolated their nuclei and the material within He called this nuclearmaterial nucleic acid Each unit of DNA contains a sugar molecule calleddeoxyriboseDeoxyriboseconsistsoffiveatomsFourcarbonatomsandoneoxygeninaringwhichbindoneofthefournucleicacidbasesadenine(A)guanine(G)cytosine(C)orthymine(T)Each[sugar+baseunit]iscalledanucleosideThe nucleosides are joined to each other by a link a phosphate

group forming long threads of hundreds of thousands of subunits Thesesubunits[sugar+base+phosphatelink]arecallednucleotides

RNA is essentially the same asDNA except that it has another oxygenatomincludedineachribosesugarmoleculeSoaDNAsugarunitisactuallyan RNA sugar unit without one oxygen which is why it has the nameldquodeoxyriboserdquo

Miescher thought nucleic acid could only have a simple and boringfunction sinceDNAmolecules are composedof long stringsofnucleotidesthatarealmostidenticalapartfromhavingoneoffourpossiblebasesattachedHe thought that it might serve as a kind of scaffolding that just sat in thenucleusandsupportedmoreldquointerestingrdquomoleculessuchasproteinsProteinsinturnaremadeupofstringsofaminoacidsofwhichtherearetwenty-twodifferentvarieties

NothingcouldbefurtherfromthetruthOneachriboseordeoxyriboseunitanotherchemicalisaddedInthecase

ofDNAtheseareoneofthefourbasesadenosine(A)guanine(G)cytosine(C) and thymine (T)When JamesWatson andFrancisCrick elucidated thedouble helix crystal structure of DNA in 1953 they found that these fourbasesldquopartneruprdquo

TheUnknownHeroineBehindWatsonandCrick

RosalindFranklin(1920ndash1958)wasaBritishbiophysicistandgraduateofCambridgeUniversitywho used X-rays while working at the Medical Research Council Biophysics Unit at KingrsquosCollege London to define crystal structures It was her data on the structure of DNA and inparticular photograph 51 that Watson and Crick relied on without her knowledge or muchrecognitiontoworkoutthestructureofDNAHerarticleswhichoutlinedthestructureofDNAbased on her ownworkwere eventually published in the same issue ofNature asWatson andCrickrsquosbetterknownpaperFranklinCrickWatsonandMauriceWilkinswerekeentobeatLinusPaulingtoestablishtheactualstructureofDNAafterPaulinghadproposedanincorrectstructureforDNA

When the fourbasesldquopartneruprdquo theyareboundweaklybyhydrogenbonds A binds to T and vice versa C binds to G and again vice versaAdenineandguanineareexamplesofpurinebasesandcytosineandthymineare pyrimidines (Figure 31a) In DNA a long strand of the linkeddeoxyribosesugarsbearingoneofthefourbasesoneachsugarmoleculenowcalleddeoxyribonucleicacidisldquozippeduprdquobypairingwithacomplementarychainofnucleotides(Figure31b)ontheotherDNAstrandTheselongpaired

chainsofcomplementarynucleotides(iedoublestrands)arethenwoundintoahelix(Figure31c) foldedandballedup tomakeupgeneswhich in turnarelinkedtogetheronchromosomes

Figure31aShowinghowthefourbasespairupbyhydrogenbondingThepyrimidinebaseThymine(T)partnerswiththepurinebaseAdenine(A)andthepyrimidinebaseCytosine(C)partnersupwiththepurinebaseGuanine(G)ThesebasesarethenattachedtothedeoxyribosesugarringsofDNAortheribosesugarringsofRNAThesugarsarejoinedtogetherbyphosphatelinkagestoformphosphate-deoxyriboseorphosphate-ribosebackbonesaccordingly(Figure31b)EachdeoxyriboseplusbaseplusphosphateunitiscalledanucleotideThenucleotidesarejoinedtogetherintoverylongsequencesoftenoveramillionunitslongInthecaseofDNAthesethenlineupagainsttheircomplementarypartnerstoformadoublestrandandarethenwoundintothecharacteristicdoublehelixformation(Figure31c)

Figure31bThephosphate-deoxyribosebackboneofDNA

Figure31cThecharacteristicdoublestrandformationofDNAwoundintoadoublehelixwhichisthenfurtherfoldedandtwisted

DNAisagreatwaytostoreandtransmitinformationinthiscasegeneticinformation But more than that scientists have even been able to storeinformationinstrandsofDNAthatyouorIwouldthinkmorelikelybelongonacomputerRecentlyateamledbyProfessorGeorgeChurchatHarvardreported in themagazineScience that theyhadstoreda53000wordbookincluding eleven images and a computer program (527 megabits ofinformation)inastrandofDNAThescientistswentontosuggestthatasthetechnology improveswemight seeDNA replacing conventional computersOnegramofDNAabouttheweightofapinchofsaltcanstore455billiongigabytesof informationThat is thesameamountof information that todaywouldbestoredononehundredbillionDVDs

ThereareadvantagesofstoringinformationinDNAasallspeciesonthisplanethavefoundItiseasilycopiedandpassedontothenextgenerationItcanstillbereadthousandsofyearslateraswearenowdoingwiththeDNAfromEgyptianmummiesAndthewaytoreadDNAdoesnotchangeunlikethe rapidly changing digital storage media that make todayrsquos informationtechnologybreakthroughstomorrowrsquosrelicsforthemuseumorrecycling

ChromosomesWe humans have 46 chromosomes consisting of 22 pairs and two sex

chromosomes(XXinthecaseofwomenandXYinthecaseofmen)(Figure32)Onthesechromosomeswehaveapproximately25000genesintotal

Figure32The22pairsandtwosexchromosomes(inthiscasefromamanndashwiththecharacteristicshortYsexchromosome)ofahuman

Most of our genes regulate the production of a protein of somedescription with different genes in different parts of the body being moreactive than elsewhere Think of it as the information the blueprint in thebooks(genes)beingtakenoutofthelibraryandsenttoahouseorfactory(theribosome)Theribosomesaresmallorganelleswithinthesamecitythecellwhere this information can then be read understood and used to producesomethingndashinthiscaseaproteinTheribosomeisaprotein-makingmachineintheouterpartofthecell

Eachandeverynucleatedcell in thebodyhas thesame librarywith thesame25000booksthegenesButdifferenttypesofcellswillbeprogrammedtoproducedifferentproteinsbytakingtheinformationfromdifferentbooksinthe library The book in this case the gene does not leave the library thenucleus but the information the blueprint it contains is conveyed to theribosome factory in the cell cytoplasm by a messenger a copy of theinformationThatcopyismessengerRNAmRNA

RNA

MessengerRNAiscleverlymadeWhenthedoublestrandofDNAinthenucleus unzips new sequences of complementary material bind to theexposed single strand of DNA and are then peeled off (transcribed) asprecursor messenger RNA (pre-mRNA) (Figure 33) In the process RNAincorporates the pyrimidine base Uracil (U) instead of the Thymine that isfound inDNAThe formedRNAunits [ribose+base+phosphate linkage]arealsocallednucleotides

However there is much information within these DNA strands that iscurrently considered unnecessary Think of the ldquobookrdquo (the gene) beingcomposedofmanychaptersInthecaseofthelargestgeneinthebodythatfor dystrophin the gene consists of 2400000000 nucleotide units Thesenucleotides are split up into ldquosectionsrdquo (chapters) of exons and introns Fordystrophin thereare79exons(accountingfor06of thegenersquossequence)and78introns(accountingfortheremaining994ofthe24billionlettersofthegene)Only theexonsareneeded for the information theycontain tobetranslated into thedystrophinproteinby the ribosomeMostofdystrophinrsquosexons are relatively small at between 23 and 269 nucleotides in lengthcomparedtotheaveragesizeofthedystrophinintronswhichareover26000nucleotideslong

Alltheinformationrequiredbythemusclecellformanufacturingthefinalproteindystrophiniscontainedinthe79exonsndashjust06ofthedystrophingene

Figure33SimplediagramoftranscriptioninitiationRNApolymeraseisanenzymethatunzipsthedoublestrandofDNAandmovesdownthegene(blackcodingthread)

AsthedoublestrandldquounzipsrdquothepolymeraseassemblescomplementaryRNAunitswhichgraduallylengthenasthegeneunzipsandthenzipsbackupagain

EventuallythegenehasgeneratedafullcomplementaryRNAstrandandtranscriptionterminatesThenewpremessengerRNAiscompletedandreleasedTheDNAdoublestrandldquozipsrdquobackupandtheRNApolymeraseisfreetoworkonanothergene

The introns by and large carry information that is not needed by thefactory in the cell cytoplasm so they can be removed as the pre-mRNA istidiedupandconvertedintousefulmRNAWenowknowthatthisprocessofldquotidyinguprdquowhichiscalledsplicingisacomplexprocessperformedinthenucleusandmayinfactleadtodifferentldquoalternativerdquoblueprintsbeingsenttothedifferentcellularfactories

The Encyclopedia of DNA Elements (ENCODE) consortium of 442researcherslaunchedbytheUSNationalHumanGenomeResearchInstitutein2003 hasbeen examining the99ofDNA thatmakeup the introns InSeptember 2012 the scientists published 30 papers simultaneously in threemajorscientificjournalsaremarkablefeatonitsownInthosepapersmanycommon diseases such as diabetes obesity heart disease cancer andAlzheimerrsquosdementiatheonesthatarenotcausedbyidentifiedsinglegenesare now thought to be caused by genes being switched on or off by theinformationcontainedintheintronsButthestoryisonlyjustbeginningtobeunraveledWe have a longway to go to fully understandwhat 99of our

DNAdoesandhowAwholenewbranchofmedicineandrangeofpossiblenewdrugsisnowemergingthatwilltargettheseswitchesthatmaytriggeraliver cell to stop absorbing sugar and so diabetes develops or the switchwhichoncethrowninalungcellwillturnitintoacancercell

It used to be thought that one gene codes for one protein but we nowknowthatthereareroughly25000genesbutapproximately150000proteinsThuseachgeneonaveragecontainstheblueprintforsixproteinsHow

Theprocessoftidyingupcansometimesleadtothemessage(inmRNA)beingput together in differentways Imagine that thebook in the library istakenoff theshelfand thecoverandbindingareremoved thenon-essentialinformation(theintrons)isdiscarded(moreaboutthatlater)andthenacopyofthenecessaryinformationisboundintoamuchmuchslimmerbookThusthe book copy (mRNA) that will leave the library (nucleus) only containsimportantinstructionssothatwhenitreachesthefactory(theribosomeinthecell cytoplasm) the various blueprints are still recognized and can still beused to build the finished products (the different proteins) The introns thathave been discarded contained no useful information at all or so it wasthought but all of the exons do contain useful information HoweversometimesoneexonormoreismissingfromthefinalmRNAthatmakesittotheribosomeandsomeoftheusefulinstructionismissingAdifferentfinalproteinproductcanstill sometimesbemadeandmaywork for thecellandthebody

If the ldquobook copyrdquo (mRNA) was the blueprint for a car (protein) thenconsiderthattherewouldbechapters(exons)onhowmanywheelsithadaswell as a chapteronhowmanyseats ithad Itwouldhaveachapteron theinterior finish another on the size of the engine what color it should bewhere and how the alarm is set up etc All of these chapters would benecessary to produce the car as sold in the showroom functioning to themanufacturerrsquos specifications However if the chapter on the alarm wasmissing thecarwouldstillwork it justwouldnrsquothaveanalarmOr thecarmightbeadifferentcolororlackoneormorecoatsofpaintThecarmightrustquickerbutitwouldstillbedrivable

Thus 25000 genes (books in the library) may each generate manydifferentmolecules ofmRNA (copies of thinned down books consisting ofimportantinformation-exons)OnaverageeachgeneinourbodiesgeneratessixdifferentmRNAmolecules that leave the library (nucleus)andgo to the

factory(ribosome)wheretheywillgeneratesixtimesthenumberofproteinscompared to the number of original genes This process first described in1977iscalledalternativesplicing

AlternativesplicingWenowknowthat95ofourgenesthosethatarecomposedofmultiple

exons are alternatively spliced This happens across all eukaryote speciesEukaryote species are plants and animals where the cell has a nucleus andotherdiscretestructuresasopposedtoprokaryotesbacteriawherethecellsdo not have these discrete structures The current record for most mRNAvariants being generated from a single gene comes from the fruit flyDrosophila melanogaster The humble fruit fly has a geneDscam whichgeneratesmorethan38000splicevariants

The fruit fly has long been used to study genetics In this interestinginsect alternative splicing is required to produce offspring of each genderPre-mRNAs from theDmelanogaster genedsx contain 6 exons Inmalesexons1235and6(skippingexon4)arejoinedtoformthemRNAwhichcodes for a regulatory protein required for male development In femalesexons 1 2 3 and 4 are joined (leaving out exons 5 and 6) The resultingmRNA is a regulatory protein required for female development (Also seeFigure41)

Continuingtheanalogyofthebook(orblueprint)endingupatthefactory(ribosome) out in the cell cytoplasm (the city) The book is composed ofwordsEachwordiscomposedofthreelettersbutthewordsallmakesenseEach letter is a nucleotide and eachword (of three nucleotides) is a codonCodonsarebundledtogetherndashsometimesonlyafewothertimesthousandsto formchapters in thebookEachchapter is anexonTheblueprintmakessenseifthewordsinachaptermakesenseHoweversometimesthebookisdamaged and a chapter severalwords or even a single letter in aword ismissingTheprocessoftidyingupthebookintoamanageablecopyldquosplicesrdquothe chapters of useful information (exons) together and removes the non-essential information (introns) by arranging everyword (codon)with threeletters (nucleotides) into sentences The mRNA strand (the copy of theblueprint)canthenbetranslatedbythefactory(theribosome)intoaprotein

However if a single letter ismissing during the transcription process aletteristakenfromthenextwordtocompletethepreviouswordThefactory

canonlyreadwordsinsequenceEachwordhastobeofthreelettersduringthisldquosplicingrdquoforthesentence(andhencethechapterandthusthebook)tomakesense If thewordsarespliced incorrectly thebookcannotbeldquoreadrdquoandthefactory(ribosome)willnotbeabletomaketheprotein

Considerthisexample

thebigredfoxranfarandsawthedogandcathittheman

This informationcanbe readandunderstoodbut if theldquogrdquo ismissed inbig thenldquorrdquo isusedfromredtocomplete thecodonframeshiftoccursandthesentencenolongermakesanysense

thebiredfoxranfarandsawthedogandcathittheman

InpracticethefactorywillstartoffreadingthemessageItwillbeabletofollow the instructions for ldquotherdquo but none of the other codons will makesense So the rest of the sentence will not be interpreted or worse maygenerate bad proteins This situation occurs in some perhaps many raregenetically determined diseases It is estimated that 70 of neurologicaldiseasesareassociatedwithsomeformofupsetintheregulationofalternativesplicing

Thereare64possiblecodonsof three lettersgeneratedbythefourbasesACGandTAnyoneof the fourbasescanbeplaced inanyof the threepositionsinthecodonthusthenumberofpossibleoptionsis4x4x4whichequals64

ProteiniscomposedofchainsofaminoacidsThereare21aminoacidsofwhich twenty are coded for by one or more of the 64 individual possiblecodonsThatiswhyitisvitalforthethreelettersinacodontobefaithfullyreproduced If adifferent sequenceof three letters ismade eithernoaminoacidwillbecodedfororthewrongoneisincludedThismayturntheproteinfromaldquogoodrdquoproteinintoaldquobadrdquoproteinasinthecaseofsicklecellanemiawhere a single nucleotidemutation leads to the hydrophobic (water hating)amino acid valine replacing the hydrophilic (water loving) amino acidglutamicacidononeofthehemoglobinproteinchains

SickleCellAnemia

Sicklecellanemiaisprevalentinthetropicsandespeciallyinsub-SaharanAfricawhereroughly

onethirdofindigenouspeoplecarrytherecessivegeneInAfricacarryingonecopyofthegene(asitisrecessivethediseaseonlyoccursifbothparentscontributeonecopyofthesicklegene)hasoverthecenturiesprovidedsomeprotectionagainstmalariaprovidingacompensatorybenefitThis survivaladvantagehasallowed thisotherwiseunhealthy trait to remainwidelyprevalent inAfricawheremalariatooisprevalent

Thoseunluckyenoughtobehomozygousandtohavetwocopiesofthesicklegenefindtheirredblood cells become rigid and distorted under themicroscope acquiring the characteristic sickleshape and they get stuck in the small capillaries robbing downstream tissue of oxygen gettingdestroyedintheprocessleadingtoanemia

Healthy red blood cells generally survive 90 to 120 days before being replaced but sickle cellsonlylast10to20daysandthebonemarrowcannotkeeppacewiththisrapidturnoverIn1956VernonIngram(1924ndash2006)aprofessorofbiologyattheMassachusettsInstituteofTechnologyalongwithJohnHuntandAntonyStrettonfirstreportedthatsicklecellanemiawascausedbyasingleaminoacidsubstitutioninthehemoglobinmoleculeThisledtoIngrambeingcreditedastheldquoFatherofMolecularMedicinerdquo

SummaryThe city (cell) has a library (nucleus) containing 46 shelves

(chromosomes)ofbooks (genes)madeoforiginal paper (DNA)Eachbook(gene)inthelibrarycontainsmanychaptersofusefulinformation(exons)andunnecessary information(intronsndashorat leastwecurrentlybelieve thatmostintronsdonotprovideuseful information)Eachchapter(exon)iscomposedofmanywords(codons)ofthreeletters(nucleotides)Thefactoryinthecelldoesnotneed theoriginalbook from the libraryndash just a copyof theusefulinformation(containedintheexons)fromthebookAttheshelvesthebookistakentopiecesandtheoriginalcodonsof3letters(ATCandG)matchedup with complementary letters (U A G and C) of a growing strand ofprecursormRNAThiswholecopyof thebookaspre-mRNA issent toberebound (ldquosplicedrdquo) by the librarian (the ldquospliceosomerdquo) where the non-essentialinformationisdiscardedandtheremainingchaptersareldquoreboundrdquoasthematuremRNAwhich then leaves the libraryandheads to the factory Ihave tried to simplify this complex process while describing with somelicensethemainstepsEachyearmoreisunderstoodabouttheprocessthevarioussignalingandregulatingchemicalsandevenmorequestionsarise

How does the spliceosome form and why Somewhere someone isbeaveringawayonthatveryquestion

Whatcontrolstherateatwhichtheseprocessesprogresstherebindingforinstance

WhatcontrolstherateatwhichthebooksaretakenofftheshelvesHow is the speed at which the newly bound books leave the library

regulatedIntenseinvestigationistryingtorevealthemechanismsthatperformthese

importantcontrolsWe do know now that there are other types of RNA microRNA and

regulatory RNA which seem to control some of these steps The intronspreviously thought to contain no vital information may be critical andinvolved in theseprocessesAs sciencephrases the important questions andtheanswersarediscoveredcorrectingmisconceptionsnewquestionswillberaised

Right now we are already beginning to develop synthetic oligomerswhich will camouflage small pieces of the messenger RNA in some casestriggeringalternativesplicingand thusgivingusmedicines todefysomeofourpreviouslyuntreatablediseases

W

ChapterFive

TheEndofHereditaryRareDisease

henIwasafamilydoctorback inEnglandin the80sand90s Ihadatleastonepatientwithararediseaseonmylistofthreethousandpatients

AnthonyMichaels lived with his devoted mother in a small modern two-bedroom house on the other side of town Anthony had cystic fibrosis Inthosedayshehadalifeexpectancyofonlysixteenyears

Anthony was in and out of the big regional hospital in Birmingham(Englandrsquos second biggest city) every few weeks and indeed he had ascheduleofadmissionsprearrangedsothathecouldreceiveintensecoursesofintravenousantibioticsandphysical therapy tohelpclear thestickyplugsofmucusblockinghisairwaysthatischaracteristicofcysticfibrosis

Despite these regular admissionsAnthonywould frequently succumb tosomenew chest infection his breathingwould deteriorate his coughwouldget worse and his temperature would climb He would be rushed into thehospitalforemergencycoursesofhisusualtreatmentofpowerfulintravenousantibioticsandtobeputonabreathingmachine

When Iwould see him a fewweeks later hewould look emaciated butbothheandhismumwouldbe jokingandplayfully teasingmedespite theknowledgethatasateenagerhewasnowonlyexpectedtoliveanotheryearortwo

OnedayIwasaskedbythespecialistpediatricpulmonologistat thebigregional center who looked after about thirty children with CF whether IwouldgiveAnthonyldquohomeintravenousantibiotictherapyrdquoWhatthatmeantwas that I (or one of the competent and experienced district nurses) wouldhavetogointotheMichaelsrsquohomeputanintravenouslinewithapowerfulantibiotic(gentamicinifmymemoryiscorrect)intooneofAnthonyrsquosfragileveinsandafteraperiodofobservationtoensureallwaswellremovetheIVaccessandtidyup

In those days there were no inhaled antibiotics and although Anthony

dailytookmanycapsulesofthevitalpancreaticenzymesupplementsthatherequiredtherewasnoeffectivetreatmenttokeepthelungsclearofthenastybacteriaespeciallyPseudomonasaeruginosawhichhethereforeharboredinlowlevelsevenonthebestofdaysOnbaddaysthenumberofthesenastybugs would surge and down he would go with the next chest infectionInevitablyadaywouldcomewhenthebacteriawouldbecomeresistanttothelifesavinghighdosesofgentamicinhereceived

IagreedtoprovideldquodomiciliaryIVantibioticcarerdquoforAnthonyalthoughithadnotbeenldquoapprovedrdquoorbecomestandardpracticeatthattimeAlreadythe expensive drugs I prescribed to keepAnthony alive had attracted someconcernfromthelocalhealthcareadministratorsandfewotherGPswithintheWestMidlandsregioncoveringalargepartofcentralEnglandhadagreedtogiveIVantibioticstotheirCFpatientsathomepreferringthemtojourneyintotheregionalcenterfortheirregularweeklycourse

Over the years Anthony had a series of setbacks and he had severaloperationsforsquintsunrelatedtohisbreathinganddigestiveproblemsIleftmypracticebutmyguessisthathewouldsurelyhavebeenoneofthosetoreceive the life enhancing cycling inhaled tobramycin antibiotic (TOBI acousin of gentamicin) by nebulizer which contributed to life expectancydoubling to 32 years Hewould probably have been eventually listed for alungordoublelungorevenaheart-lungtransplant

For several years I worked as a drug development physician at ChironCorporation (acquiredbyNovartis in2005)whichwasworkingwithTOBIforCFandanotherpulmonarydiseasebronchiectasisChironthenmovedontowork on a project using a drug to prevent lung transplant rejection Thememory of Anthonyrsquos cheeky grin and bubbly sense of humor alwaysremindedmeofwhatphysiciansinthepharmaceutical industryaretryingtoachieve thedevelopmentofdrugsthatwilladdyearsofgoodqualitylife topeoplelikeAnthony

IbelievethatAnthonydidindeedreceiveaheart-lungtransplantbutthathedidnotlongsurvivethatprocedure

Anthonywasoneofthepatientswhosehumorandfortitudeinthefaceofadversityandwith littlehopeof livinganormal lifespanorqualityof lifeinspiredmeasadoctorAtleastafirmdiagnosishadbeenmadeandalthoughtherewas thennodefinitive treatmenthismother andhewere toldwhat toexpect

I had other patients who had no diagnosis at all They had strangeconditions and were repeatedly examined by top specialists who agreedsomething was amiss but couldnrsquot tell what it was In this new era ofgenomicsmany of these rare diseases alasmost still lacking any effectivetreatment can at least be accurately diagnosed and the specific geneticmutationidentifiedAndmanymoreoftheserarediseaseswillsoonnodoubtberevealed

Currentlyaboutseventhousandrarediseaseshavebeencharacterizedandthe faulty gene isolated There are estimated to be at least another seventhousandwhichhavenotyethadacausativegeneticmutationidentifiedandthusareuntreatablebutgraduallythesediseaseswillbeunderstood

Mostof these raregeneticdisorders areonlydiagnosedonce thepatienthasdevelopedsymptomsofthedisorderbringinghimorhertotheattentionof doctorsAnd because these diseases are due to faulty genes the child isdestinedtodevelopthediseasefromthemomenttheyareconceivedliterallywhenthefatherrsquosspermmeetsthemotherrsquosegg

ThesegeneticdiseasesareunlikelyevertobefullypreventablealthoughmanycanbescreenedforinearlypregnancywiththeoptionfortheparentstoendthepregnancyofanaffectedembryoThenextstepforthosebabiesistodevelopeffectivetreatmentsfortheserareandoftenlethaldiseases

IbelievethatoligomersmaybeapotentialanswerformanyoftheseItooamaparentaffectedbyararediseaseMulticysticdysplastickidney

(MCDK) is a rare disease occurring in one in roughly 4300 births Babiesborn with one dysplastic kidney can survive well on the remaining normalkidneyTenweeksbeforehewasbornoneofmyownsonswasdetectedwiththis raredefectUnfortunatelyheappeared tohaveablockageaffectinghisotherkidneymeaningneitherofhiskidneyswouldworkWithinhoursofhisbirthhewassubjecttoanoperationtotryandbypasshisblockedkidneybutalastheoperationfailedHewasbroughtfromtheoperatingtheatertomeandIwasgiventhenewsthathewasexpectedtodiewithinthenexttwenty-fourhoursIwaslefttocuddlethislittlebundleinahospitalcubiclemilesawayfromwherehismotherwasrecoveringfromgivingbirthtohimAgainsttheodds he survived the day the night and then the next day After severaloperations to re-plumb his solitary working but now damaged kidney andthen remove his non-functioning one he survived childhood with bloodpressuretreatmenttreatmentforthesideeffectsofthattreatmentandvarious

supplementsSoyesIknowfrompersonalexperiencehowrarediseasecanaffectafamily

There is no single widely accepted definition for rare diseases Somedefinitions rely solely on the number of people livingwith a diseaseOtherdefinitions includeother factors suchas the lackof adequate treatment theseverity of the disease or a lack of resources to care for the patient Somepeoplepreferthetermorphandiseaseanduseitasasynonymforrarediseasesuch as the European Organization for Rare Diseases (EURORDIS) whichcombinesboth rarediseasesandneglecteddiseases (thosewithno treatmentavailable)intoalargercategoryofldquoorphandiseasesrdquo

TheorphandrugmovementbeganintheUSoverthirtyyearsagoItwasthemovingforcebehindtheOrphanDrugAct(ODA)of1983afederallawdesigned to encourage research into rare diseases and possible cures TheODAincludesbothrarediseasesandanynonrarediseasesldquoforwhichthereisno reasonable expectation that the cost of developing and making a drugavailableintheUSforsuchdiseaseorconditionwill[be]recoveredfromitssalesintheUSrdquoasorphandiseasesSince1983morethan2200drugshaveentered the research pipeline and more than 360 have completed theirdevelopment and been approved for marketing Currently orphan productsaccountforaboutonethirdofallNewMolecularEntitiesbeingapproved

The subsequent Rare Disease Act of 2002 defines rare disease strictlyaccordingtoprevalencespecificallyldquoanydiseaseorconditionthataffectslessthan 200000 persons in the United Statesrdquo or about one in 1500 peoplePrevalence is defined as the total number of cases of the disease in thepopulation at a given time or the total number of cases in the populationdivided by the number of individuals in the population It is used as anestimateofhowcommonadiseaseiswithinapopulationatacertainpointintime This should not be confused with incidence (the number of newdiagnoses in a given year) which is used to describe the impact of rarediseases

TheincreasedregulatoryattentionaffordedtorarediseasesledtotheUSFood and Drug Administration (FDA)rsquos Center for Drug Evaluation andResearch (CDER) establishing a Rare Disease Program in February 2010with its ownAssociateDirector of RareDiseases (ADRD) reporting to theDirector of theOffice ofNewDrugs (OND)This new teamwas given thegoalof facilitatingandsupporting the researchdevelopment regulationand

approvalofdrugsforthetreatmentofraredisordersandwastocomplementtheworkofFDArsquosOfficeofOrphanProductDevelopment(OOPD)ItwouldbecomethefocalpointofcontactatFDAforRareDiseasestakeholderssuchascompaniesdevelopingsmallmoleculedrugsoligomersandbiologicsandpatient advocacy organizations The rare disease program team wouldfacilitate interactions with CDER and their sister Center of BiologicEvaluationandResearch(CBER)TheADRDwouldhelpdrugdevelopmentcompanies navigate complex regulatory requirements and the increasinglyintricate bureaucracy that is the FDA There is still much to do Currentlyonlyabout twohundredof theseven thousandcharacterizedrarediseases intheUShaveanapprovedtreatmentavailable

Ihaveworkedonseveralrarediseaseprogramsduringmytwentyyearsasaphysicianinthepharmaceuticalindustryandlednumerousinteractionswithregulators in the US (FDA) and Europe (European Medicines Agency orEMA) I am pleased that in some cases the new drug applications that Iworked on have already been submitted and approved I suspect that theincreasedFDAfocusonrarediseaseswill indeedleadtofasterdevelopmentprograms and more approval for ground-breaking pharmaceutical productsandhopefullyoligomersintheyearsahead

The key to understanding FDA structure roles and responsibility as itpertains to rare disease is to understand the complementary but distinctseparationbetweentheOfficeofOrphanMedicinalProducts(OOPD)andtheOffice ofNewDrugs (OND)rsquosRareDiseaseProgram (RDP)which canbesummarizedthisway(Figure51)

OOPD ONDRDPAdministratestheODAbullDesignationsbullExclusivitybullOrphangrants

FacilitatescommunicationwithinCDERONDreviewdivisions

DeviceprogramsFocusesoncomplexregulatoryrequirementsforINDsNDAsandBLAs

Pediatricfocus Developspolicyproceduresandadviceforrarediseaseclinicaldevelopmentprograms

Strongadvocacyworkwithrarediseasestakeholders

CommonareascoordinatecommunicationacrossFDAcentersandoffices

CommonareascoordinatecommunicationacrossFDAcentersandofficesandwithoutsidestakeholdersenhancerarediseaseinformationavailableonFDAwebsite

ODA=OrphanDrugAct IND=InvestigationalNewDrug (the application to theFDA to allowinitial human testing) NDA = New Drug Application (the enormous dossier containing all theresearch animal testing clinical studies manufacturing and quality testing that the FDA reviewspriortoapprovingtheproductsuitableformarketing)BLA=BiologicLicenseApplication(theNDAforbiologicproducts)

Figure51ThesimilaritiesanddifferencesbetweentheFDArsquosOfficeofOrphanMedicinalProducts(OOPD)andtheOfficeofNewDrugrsquosRareDiseaseProgram(ONDRDP)

Further evidence of the increasing FDA interest in rare diseases isprovidedbythesevenrarediseaseapprovals(threedrugsandfourbiologics)inthefirstninemonthsof2010(AppendixB)ofatotalof17NewMolecularEntities(NMEs)approved(tendrugsasNDAsandsevenbiologicsasBLAs)

InJapanthelegaldefinitionofararediseaseisonethataffectsfewerthan50000 patients in Japan or about one in 2500 people The Europeandefinition of a rare disease is a life-threatening or chronically debilitatingdiseasethatisofsuchlowprevalencethatspecialcombinedeffortsareneededtoaddressitThetermldquolowprevalencerdquoislaterdefinedasgenerallymeaningfewer thanone in2000peopleconsistentwith theEuropeanCommissionrsquosdefinition of rare Diseases that are statistically rare but not also life-threatening chronically debilitating or inadequately treated are excludedfrom their definition The definitions used in the medical literature and bynationalhealthplansaresimilarlydividedwithdefinitionsrangingfromonein a thousand to one in two hundred thousand The Global Genes Projectestimatestherearesome350millionpeopleworldwidecurrentlyaffectedwithararedisease

Although each individual rare disease is rare by definition the sheernumber of different individual rare diseases results in approximately eightpercent of the population of the European Union being affected by a rarediseaseclose to theestimatedtenpercentofUSpatientswhoaresimilarlysuspectedofsufferingfromararedisorderMostrarediseasesaregeneticandthusarepresentthroughoutthepersonrsquosentirelifeevenifsymptomsdonotimmediately appear However many rare diseases appear early in life andabout thirty percent of children with rare diseases will die before reachingtheirfifthbirthdayRarediseasescanvaryinprevalencebetweenpopulations

soadiseasethatisrareinsomepopulationsmaybecommoninothersThisisespecially true of genetic diseases and infectious diseases An example iscystic fibrosis (CF) a genetic disease which is relatively common inCaucasianEuropeansTherecessivegeneiscarriedbyapproximatelyonein25peopleForthediseasetooccurbothcopiesofthegenemustbeaffectedThus 125 x 125 partnerships are likely to result in two carriers comingtogether (ie one in 625 partnerships) These parents have a one in fourchancethattheirchildwillreceivetherecessivegenefrombothpartnersThisgivesanincidenceofonein4x625newbirths=onein2500InAsiansCFisevenrarer

Finland has a higher prevalence of about forty rare diseases these areknowncollectivelyastheFinnishdiseaseheritageAshkenaziJewsalsohavea higher prevalence of certain rare diseases with an estimated one in fourindividualsbeingacarrierofoneofseveralgeneticconditionsincludingTay-Sachs Disease Canavan Niemann-Pick Gaucher Familial DysautonomiaBloomSyndromeFanconianemiaandMucolipidosisIV

There aremany companieswhose development programs now focus onrare or orphan diseases including Synageva BioPharma Corp (based inMassachusetts) Swedish Orphan Biovitrum Shire plc (British) Genzyme(alsobasedinMassachusettsbutrecentlyacquiredbytheFrenchgiantSanofiAventis) Lundbeck (a Danish company) and BioMarin (a small Californiabased company) Disappointing as it is that there has not been more workconductedon rarediseases in thepast it is encouraging to learnhowmuchresearch is now underway Perhaps discoveries in rare diseases will havemuchgreaterimpacttofuturehealthcarethanisexpected

Take the example of progeria Progeria is a very rare disease affectingaboutone in fourmillionpeople It is nowknown tobe causedby a singlebasechangefromaCtoaTinthemiddleoftheLaminAgene(LMNA)Thiscauses150nucleotides inexoneleven tobesplicedoutof the finalmRNAand theresultingabnormalprotein lacksfiftyaminoacidsThediseasealsoknownby its longer titleofHutchinson-Gilfordprogeria syndrome (HGPS)results in rapid aging (at about seven times the normal rate) and childrendying usually around age 12 to13 years from a heart attack bywhich timetheylookliketheyareintheireighties

Themolecularbiologybehindprogeriawascharacterizedsomeyearsagowith a buildup of a toxic protein (progerin) in the cells that led to the

premature heart disease In an experimentalmousemodel the toxic proteincouldbereducedbytreatmentwithafarnesyltransferaseinhibitor(FTI)drug(lonafarnib) and the mice survived without the premature cardiovasculardisease

Since 2007 a clinical trial has been underway in progeria to see if thesame drugwill alsowork in the human disease although the ClinicalTrialGovwebsiteforthisstudy(runbySchering-PloughandtheProgeriaResearchFoundation at Boston Childrenrsquos hospital) has not been updated sinceDecember 2007 At that time the studywas slated to complete in October2009 There are only 42 identified children in the world (from at least 15countriesndashincludingPakistanCroatiaKoreaArgentinaandVenezuela)withthis rare condition making the conduct of this clinical study incrediblychallengingHavingbeenintouchwiththeProgeriaResearchFoundationinmid 2012 I understand that the results from this study will be publicallyreleasedsoonIhopetheyarepositive

Inaddition ithasbeen found that the toxicproteindoesbuildup in thecells of the elderly so perhaps a better understanding of the rare diseaseprogeriawillhavefutureimportant implicationsforagingingeneralMaybeintimeitwillbepossibletoskipthepointmutationintheprecursormRNAwithanoligomerandcreateadifferentmRNAthatwillbemuchclosertothenormal message with most of the 150 missing nucleotides restored Thiscould conceivably prevent the buildup of the abnormal progerin and thedevastatingdiseaseprogeria

William Harvey was best known for determining how the circulatorysystem worked but in 1657 he remarked in a letter about rare diseasesldquoNature isnowhereaccustomedmoreopenly todisplayher secretmysteriesthan in cases where she shows tracings of her workings beside the beatenpathnor is thereanybetterway toadvance theproperpracticeofmedicinethattogiveourmindstothediscoveryoftheusuallawofNaturebycarefulinvestigation of cases of rare forms of diseases For it has been found inalmost all things that what they contain of useful or applicable nature ishardlyperceivedunlesswearedeprivedofthemortheybecomederangedinsomewayrdquo

Manyinfectiousdiseasesareprevalentinagivengeographicareabutrareeverywhere else usually limited by the distribution of specific climaticconditions or certain animals required for their life cycle or both Other

diseases such as many rare forms of cancer have no apparent pattern ofdistributionbutaresimplyrareTheclassificationofotherconditionsdependsin part on the population being studiedAll forms of cancer in children aregenerally considered rare because so few children develop cancer but thesamecancerinadultsmaybemorecommonWithasinglediagnosedpatientonly ribose-5-phosphate isomerase deficiency is presently considered therarestgeneticdiseaseThedistributionofdiseaseareasthatweretargetedforan orphan product in development according to the FDA is provided inFigure52

Figure52DiseasecategoriestargetedbyDesignatedOrphanDrugsasapercentageoftotal(FDAdata2000-2006)

WhenIwasapracticingfamilydoctoreveryyeartherewouldbeasmallnumberofcasesamongstmy3000patientswhoeithersufferedsomestrangedisease or who joined my list with some pre-existing but undiagnosedcondition In retrospect I wonder how many of those often puzzling andfrustrating cases (for the patient their family and me) would now bediagnosablewithmoderngenesequencingtechnology

NORDInthedecadebefore1983onlytennewdrugsweredevelopedbyindustry

forrarediseasesSincetheyaffectednomorethan200000Americans theywerereceivinglittleattentionResearchdollarsandexpertisewerefocusedonthe development of blockbuster drugs (seeChapter 1) for common diseasesthatweremorelikelytorepaythehugecostsofdevelopingthemIn2010itwas estimated from publicly available data that the cost to develop a newdrugwouldexceedanastonishingonebilliondollars

Back in the early 1980s leaders of rare disease patient advocacyorganizationsrecognized that therewerecertainproblems theirpatientswithanyrarediseaseandtheirfamiliessharedItwasclearthatwhileeachdiseasemay be rare together these diseases affect millions of Americans anestimated one in ten (probably thirty million patients) As a result theycollectively campaigned calling for national legislation to encourage thedevelopment of treatments for rare diseasesThe resultwas the 1983ODAandthepatientadvocacyleaderswhohadbroughtnationalrecognitiontotheproblemfoundedtheNationalOrganizationforRareDisorders(NORD)asanumbrellaorganizationtorepresenttherarediseasecommunity

NORD a charitable organization is a unique federation of over 130voluntary health organizations that is committed to the identificationtreatment and cure of rare disorders through programs of educationadvocacyresearchandservice

Intheorymanyraregeneticallydetermineddiseasesthathavepreviouslybeenuntreatablemaynowbeamenabletooneormoreoftheoligomerseitherinclinicaldevelopmentor inearlypreclinicaldevelopmentndashoreveninstillearlierresearch

There are numerous examples of oligomers already now in the clinicalphaseoftheirdevelopmentforrarediseaseie theyarenowbeingtestedinhumansThediseasesforwhichtheyaretargetedareprovidedinFigure53AnupdateonthesevariousprogramsandmanyothermorecommondiseaseprogramswasprovidedbythesponsoringcompaniesataUSmeetinghostedbytheFDAandtheDrugInformationAssociationinApril2012

asofOctober2012AlnylamhaspartneredwithGenzyme todevelop thisdrug forJapaneseandAsia-Pacificmarkets

Figure53Oligomerscurrentlyinclinicaldevelopmentforrarediseaseindications

The companies developing new drugs for rare diseases benefit from theadvocacy and various services NORD (and its European and Canadiancounterparts)providesaimedatcomplementingthem

bull Information about diseases and referrals to patient organizations(through their website at httpwwwrarediseasesorgraredisease-informationrarediseases)

bull Patient assistance programs Since 1987NORD has helped patientsreceivedrugsthatcouldsaveorsustaintheirlifeTheyalsohelpwiththecostofinsuranceco-paymentfeesdiagnostictestsandeventravelexpensessopatientscanseedoctorswhospecializeinaparticularraredisease

bullResearchgrantsandfellowshipsbullAdvocacyonpublicpolicyissuesbull Help in forming organizations and mentoring for patient advocacy

groups

NORD in July 2011 had information about 15 clinical studies on itswebsiteforsuchdiverserareconditionsasCraniosynostosis(inCalifornia)Ehlers-Danlos Syndrome Type IV (EDS type IV) (in Washington)Hirschsprung Disease (at Johns Hopkins University) and WegenerrsquosGranulomatosis (in Toronto Canada) In addition NORD produces regularnewsletters for patients and their families and continues to stimulatecongressionalfocusonaddressingthesepreviouslyunderservedvoters

WhetherduetotheeffortsofNORDornotthegeneralpublichasbecomemoreawareoftheserarediseases

EURORDISEURORDIScanbeconsideredastheEuropeanequivalentofNORDasa

non-governmental patient-driven alliance representing more than 479 raredisease patient organizations in over 45 European countries which wasconceivedforsimilarreasonsFoundedin1997itisnowadministeredby26staff based in Paris and Brussels At the end of August 2010 EURORDISsettled into newly renovated dwellings in the grounds of the HospitalBroussais Pariswith their partners in the PlateformeMaladies Rares (Rare