insight pharma-animal models sample report

7/30/2019 Insight Pharma-Animal Models Sample Report

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Animal Models for

Therapeutic Strategies

Allan B. Haberman, PhD

InsightPharmaReports.com


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Animal Models for Therapeutic Strategies

by Allan B. Haberman, PhD

Published in March 2010 by Cambridge Healthtech Institute


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Insight Pharma Reports is a division o Cambridge Healthtech Institute, a world leader inlie science inormation and analysis through conerences, research reports, and targetedpublications. Insight Pharma Reports ocus on pharmaceutical R&Dthe technologies,the companies, the markets, and the strategic business impacts. They regularly eatureinterviews with key opinion leaders; surveys o the activities, views, and plans o individualsin industry and nonprot research; and substantive assessments o technologies and markets.Managers at the top 50 pharma companies, the top 100 biopharma companies, and the top50 vendors o tools and services rely on Insight Pharma Reports as a trusted source obalanced and timely inormation.

Reae Reprs

Approaches to Reducing Phase II Attritionby Allan B. Haberman, PhD

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Animal Models for Therapeutic Strategies

by Allan B. Haberman, PhD

A Cambridge Healthtech Institute publication 2010 by Cambridge Healthtech Institute (CHI).This report cannot be duplicated without prior written permission rom CHI.

Every eort is made to ensure the accuracy o the inormation presented in Insight Pharma Reports. Much othis inormation comes rom public sources or directly rom company representatives. We do not assume anyliability or the accuracy or completeness o this inormation or or the opinions presented.

Cabid Hahch Ii, 250 First Ave., Suite 300, Needham, MA 02494Phone: 781-972-5444 Fax: 781-972-5425 www.InsightPharmaReports.com

Abu Au

Aa B. Haba, PhD, is Principal o Haberman Associates, a consulting rm specializing in science andtechnology strategy or pharmaceutical, biotechnology, and other lie science companies. He is also a Principal andFounder of the Biopharmaceutical Consortium (www.biopharmconsortium.com), an expert team formed to assistlife science companies, research groups, and emerging enterprises to identify and exploit promising breakthrough

technologies. Dr. Haberman is also the author o numerous publications on the pharmaceutical and biotechnologyindustries, their technologies and products, and on the major therapeutic areas or drug discovery and development.Formerly the associate director o the Biotechnology Engineering Center at Tuts University, he received his PhD in

biochemistry and molecular biology rom Harvard University.

For more inormation about published Insight Pharma Reports, visit www.InsightPharmaReports.com or callRose LaRaia at 781-972-5444.


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Executive Summry

Model organisms have long been a mainstay o basic and applied research in the lie sciences.Among model organisms, it is model animals that have had a central place in medicalresearch and in pharmaceutical and biotechnology company research, including drugdiscovery, preclinical studies, and toxicology. Although pharmaceutical companies have longemployed animal models based on such mammalian species as mice and rats, dogs, cats, pigs,and primates, more recently the pharmaceutical/biotechnology industry has also adoptedseveral invertebrate and lower vertebrate animal models that have emerged rom academiclaboratories. These animal models include the nematode Caenorhabditis elegans, the ruit fyDrosophila, and the zebrash. The adoption o invertebrate and zebrash animal modelsby industry has been driven by the advent o genomics, especially the nding that not onlygenes, but also pathways, tend to be conserved during evolution.

Researchers use animal models in basic research, in developing new therapeutic strategiesor treating human diseases, and in drug discovery research (including target identicationand validation, drug screening and lead optimization, and toxicity and safety screening),as well as in preclinical studies o drug saety and ecacy. The use o animal models indeveloping novel therapeutic strategies or human diseases overlaps with basic researchthat uses animal models to understand physiological and disease pathways. But its aim is toachieve knowledge o pathways and targets in a disease that leads to the development o newparadigms or discovery and development o drugs or other therapeutics. It thus also overlapswith use o animal models in drug discovery. The use o animal models in development onovel therapeutic strategies is the main emphasis o this report.

The creation o new animal models is an important part o animal-based therapeutic strategyresearch. A major reason why the pharmaceutical/biotechnology industry needs new animalmodels is because poorly predictive animal models are a major cause o drug attrition duringdevelopment. This is especially true in therapeutic areas (e.g., oncology and central nervoussystem [CNS] disease) in which animal models are the most unpredictive. Due to the poorpredictivity o many animal models, some researchers would like to work with humanmodels based on induced pluripotent stem (iPS) cells. This early stage technology mayallow researchers to develop disease models based on cells rom people with such genetically

determined diseases as spinal muscular atrophy (SMA), Parkinsons disease, Huntingtonsdisease, Duchenne and Becker muscular dystrophy, amyotrophic lateral sclerosis (ALS),


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Executive Summary

etc., which more aithully model the cellular basis o these diseases than animal models.However, most human diseases involve interactions between multiple organs and tissues.

Researchers thereore need to continue to use animal models, which are whole, livingsystems that model physiologynot just cell and molecular biology. Cellular models basedon iPS will be used in screening (which may in some cases reduce the numbers o animalsneeded in a drug discovery program) and to provide inormation on human disease pathwaysto supplement inormation derived rom studies with animal models. Inormation derivedrom cellular models, and in some cases the cells themselves, may also be used to design newanimal models to more aithully model human diseases.

The nal section o Chapter 1 discusses the issue o animal welare, which is an importantconsideration in research involving animals. The United States and various European

countries, as well as other jurisdictions (national and local), have sets o animal welareregulations. Central to these regulations is implementation o the 3Rs (Reduction,Renement, and Replacement.) Academic and corporate research organizations have beenincorporating these regulations into their research practices.

However, some types o animal research, especially research involving nonhuman primates,are particularly controversial. Moreover, animal rights activists have had their impact on thepractice o animal research, especially in Europe.

The absolutely essential need or animal research or progress in medical science andhealthcare is well proven, and researchers and the general public generally support animalresearch. However, there is an increasing concern or animal welare, including pressureor research organizations to nd ways to reduce the numbers o vertebrate animals used inresearch. There is also the increasing need or researchers and their organizations to osteropen engagement with the public and policy-makers to promote the value o animal researchand discuss animal welare issues.

Chapters 2 through 7 each ocus on a particular type o animal model. Chapters 2, 3, 4, and6 ocus on C. elegans, Drosophila, the zebrash, and the mouse, respectively. Each chapterincludes case studies o the use o each o these established animal models in developingnovel therapeutic strategies or human disease. Chapters 5 and 7 ocus on emerginganimal models or use in drug discovery and development o new therapeutic strategies,the Arican clawed toad Xenopus tropicalis (Chapter 5) and emerging mammalian animalmodels (Chapter 7). Each o these two chapters ocuses on technological developmentsnow in progress to develop tractable animal models based on these organisms or use indrug discovery research. Chapter 7, in addition to the development o model systems basedon non-rodent mammals (mainly pigs, errets, and marmosets), includes a discussion o thereemergence o the laboratory rat as an animal model. The rat, despite its important usesin physiological research, in studies involving surgery, and in other types o studies, hasbeen eclipsed by the mouse in the post-genomic era. However, it is now reemerging as

the result o new technologies (e.g., the sequencing o the rat genome and the constructiono knockout rats via various novel gene-targeting technologies) and collaborations. Some


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Animal Models for Therapeutic Strate

o these technologies are also being applied to the development o nonrodent mammalianmodels.

Chapter 8 discusses the use o computer models and translational biomarkers in helpingresearchers to more eectively move rom preclinical animal studies to human clinical trials.Pharmaceutical and biotechnology company researchers have been increasingly applyingpharmacokinetic/pharmacodynamic (PK/PD) modeling to all stages o drug development.This especially includes moving rom preclinical animal studies to human clinical trials.These models, as well as biophysical models such as those developed by Novartis andphysiological models such as those developed by Entelos, can help researchers moreeectively use animal model data in the design o clinical trials. In particular, they can helpresearchers reduce drug attrition in clinical trials due to suboptimal dosing.

Entelos virtual NOD mouse model or type 1 diabetes can be described as a virtual animalmodel. This mathematical model is made possible by the extensive studies that havebeen carried out over 30 years with the living NOD mouse, and the acceptance by thediabetes research community o the useulness o this mouse model and its relatively aithulmodeling o human disease. The virtual NOD mouse is designed to help researchers designmore eective animal studies using ewer animals, and hopeully to design more eectiveand successul clinical trials o agents designed to prevent progression to type 1 diabetes.Nevertheless, the useulness o the virtual NOD mouse in enabling researchers to discoverinnovative drugs that achieve proo o concept in clinical trials, let alone reach the market,

remains to be conrmed.

Chapter 6, which ocuses on the mouse, concludes with a discussion o the issue odeveloping more predictive animal models o drug ecacy, specically more predictivemammalian models. The two main reasons or researchers diculties in producingpredictive mouse models are 1) dierences between the mouse and humans, and 2) majorunknown factors in disease biology. These unknown factors have been revealed, for example,by studies in human genetics showing that common variants do not account or most o theheritability o disease, the discovery o the role o copy number variation and o non-codingDNA sequences in human disease determination, and the discovery in recent years o new

layers o cellular regulation based on small regulatory RNAs and epigenetics. Althoughthese actors make developing predictive animal models dicult, researchers can useanimal models to learn about unknown or poorly understood areas o disease biology. Thisis expected to lead to the development of improved animal models and the developmento new therapeutic strategies and drugs. Researchers can also bridge some o the dierencesbetween the mouse and humans by creating humanized mouse models. In some cases, othermammalian species may be better models or certain diseases (e.g., rats or cardiovasculardiseases, pigs and errets or cystic brosis) than the mouse. As discussed in Chapter 7, thereare new technologies or producing gene-modied disease models based on these mammalianspecies.

Developing animal models that are more predictive o ecacy is an iterative process. Butprogress is being made, as researchers apply new knowledge and experimental approaches


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Executive Summary

in elucidating the biology o particular diseases to creation o animal models. Researchersdeveloping new drugs for complex diseases are well advised to test drugs in more than one

animal model and in mouse strains o dierent genetic backgrounds. They should also, ipossible, employ translational ecacy and/or pharmacodynamic biomarkers to link theecacy seen in preclinical studies with clinical results.


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Tble of Contents

ChApter 1

IntRoductIon ..........................................................................................................................1

1.1. uses Me orgaisms .........................................................................................................2Basic Research ...........................................................................................................................3Developing Therapeutic Strategies ...........................................................................................4Target Evaluation ......................................................................................................................6Preclinical Studies .....................................................................................................................7

1.2. W d We nee new Aima Mes? ................................................................................9Animal Models Used in Drug Discovery and Preclinical Studies Need to be MorePredictive o Clinical Results ....................................................................................................9

Can animal models be replaced with human cellular models in drug discovery? ...........10

New Animal Models to Aid Researchers in Understanding Disease Biology andDeveloping New Therapeutic Strategies ................................................................................11

1.3. te Isse Aima Weare a Is Ees Aima Resear i drg disvera Preiia Sies ...........................................................................................................12The 3Rs ...................................................................................................................................15The Eects o Public Perception and Behavioral Research on Support o AnimalResearch ..................................................................................................................................16

ChApter 2

thE nEMAtodE CAenorHABDItIs elegAns AS A ModEl SyStEM ...................192.1. Iri .............................................................................................................................19

2.2. A C. a Me Parkiss disease ..........................................................................20

2.3. usig C. a as a Parm r drg disver a targe Ieifai viacemia Geeis Sies ....................................................................................................24

2.4. A C. a Me Spia Mse Arp.....................................................................26

2.5. csis..............................................................................................................................29


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Table of Contents

ChApter 3

thE RuIt ly DrosoPHIlA melAnogAster AS A ModEl SyStEM ................31

3.1. Iri ............................................................................................................................31

3.2. use RnAi Srees Iei drg targes i drspia ces a a nveAppra caer terap ................................................................................................32

3.3. A drspia Me r hma Gima ...............................................................................37

3.4. csis ............................................................................................................................41

ChApter 4

thE ZEbRAISh DAnIo rerIo AS A ModEl SyStEM.................................................43

4.1. Iri ............................................................................................................................43

4.2. use rwar Geei Srees i targe Ieifai i Zerafs: te case Psi Kie disease (PKd) ........................................................................................44

4.3. Zerafs Mes Meama ..............................................................................................47The Relationship between Melanocyte Development and Metastatic Melanoma ................ 48Using Melanoma Genetics to Design Zebrash Models o Melanoma .................................. 51Genes Involved in Melanocyte Development Can Synergize with Oncogenes toProduce Metastatic Melanoma ...............................................................................................53Design o Zebrash Model Systems or Use in Developing Further Understanding oMelanoma Pathobiology ........................................................................................................53

4.4. te Japaese Meaka (oyzia aip): A Emergig is Me ....................................54

4.5. Zerafs cmpaies ..............................................................................................................55Phylonix ..................................................................................................................................55Znomics ..................................................................................................................................56Evotecs Zebrash Technology Platorm .................................................................................56The Future o Zebrash Platorm Companies .........................................................................57

4.6. csis ............................................................................................................................58

ChApter 5

XenoPus troPICAlIs: An EMERGInG ModEl SyStEM ...........................................59

5.1. Iri ............................................................................................................................59

5.2. devepig Geei a Gemi ts rX. picai....................................................60

5.3. Sies wiX. picai wi Reevae hma disease.............................................62

5.4. csis..............................................................................................................................63

ChApter 6

MouSE ModEl SyStEMS ........................................................................................................656.1. Iri ............................................................................................................................65


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6.2. bakgr: cmpex diseases Are dif Me ....................................................66

6.3. cmpreesive Sraegies Imprve Mse Mes ..........................................................68

Gene Disruption Technologies, Functional Genomics, and Target Validation .................... 69Natural Variation and Quantitative Trait Loci ....................................................................... 69Chemical Mutagenesis to Create Point Mutations .................................................................71Modeling o Polygenic Traits to Improve the Predictiveness o Mouse Model Studies .........71Modeling o Copy Number Variation .....................................................................................73Humanized Mouse Models ......................................................................................................73

6.4. oer Sraegies r Imprvig Mse Me Sies: Pepig a MeigEvirmea ars ...........................................................................................................74Phenotyping ............................................................................................................................74Modeling Environmental Factors............................................................................................75

6.5. case S: A Mse Me Aism base cp nmer Variai .....................776.6. case S: A dieree ewee e Mse a hmas Ma Ae drg

disver i diaees ..........................................................................................................79

6.7. case S: usig a Imprve Mse Me Pareai caer devepnve terapei Sraegies ...............................................................................................81

6.8. csis..............................................................................................................................84

ChApter 7

EMERGInG MAMMAlIAn ModEl SyStEMS ...................................................................87

7.1. Iri .............................................................................................................................87

7.2. te Reemergee e larar Ra ..............................................................................87

7.3. Sie-diree Mageesis i Mammaia Mes oer a e Mse..........................89

7.4. Zi-iger nease Geme Eiig Pre Kk Ras ....................................90

7.5. creaig Kk Mie a Ras rm cre Spermagia Sem ces..................92

7.6. Pri trasgei Marmses ta trasmi trasgees teir osprig ......93

7.7. csis ............................................................................................................................96

ChApter 8

MoVInG RoM AnIMAl ModElS to thE clInIc ......................................................97

8.1. Meig a Simai ........................................................................................................97Computer Modeling and Simulation is Complementary to, but Cannot Replace,Animal Studies ........................................................................................................................97

8.2. cmper Meig a Simai r Mvig rm Aima Mes e cii........99Allometric Scaling: Determining the Human Equivalent Dose (HED) ................................99Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling ....................................................100Modeling and Simulation at Novartis ................................................................................. 102

Entelos ..................................................................................................................................103Entelos/American Diabetes Association virtual NOD mouse model ........................... 104


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Table of Contents

8.3. trasaia bimarkers .....................................................................................................106

8.4. csis............................................................................................................................107

ChApter 9

outlooK...................................................................................................................................109

9.1. Aima Weare Isses ..........................................................................................................110

9.2. Esaise a Emergig Aima Mes ..................................................................110

9.3. Avaages usig Ivererae Mes a e Zerafs i drg disverResear................................................................................................................................111

9.4. Aima Me Sies hep Researers lear A new Aspes disease

big ................................................................................................................................112

9.5. devepig Mre Preiive Aima Mes drg Efa ..........................................112

ChApter 10

thouGht lEAdER IntERVIEWS......................................................................................115

10.1. Aria hi, Pd, Assiae direr, Eve, Aig, oxrsire, uK................115

10.2. davie M, dVM, crprae Seir Vie Presie, cares River,Wimig, MA ..................................................................................................................121

10.3. bria W. Sper, Pd, Resear Afiaes Prgram, Sieif liais, te Jakslarar, bar harr, ME ...............................................................................................124

10.4. A Ser, Pd, direr biemisr, Sexis, Resear triage Park, nc ..130

ChApter 11

InSIGht PhARMA REPoRtS AnIMAl ModElS SuRVEy: JAnuARy 2010 ........135

Qesi 1. Pease assi r rgaizai.............................................................................135

Qesi 2. Wa aspe(s) e rg evepme press wrk i? .....................136

Qesi 3. Wa ass(es) rgs wrk ? ..............................................................136

Qesi 4. d wrk ire wi aima mes? ............................................................137

Qesi 5. I aswere es qesi 4, i wa aspe rg evepme wrk wi aima mes? ..................................................................................................137

Qesi 6. Wa pes aima mes es r mpa se i-se? ........................138

Qesi 7. Wa pes aima mes are se i sies a r mpa sres cRos? ..................................................................................................................................138

Qesi 8. d agree a pr preiive aima mes ave ee a majr reas re w privi rg evepme? ........................................................................139

Qesi 9. has ere ee a imprveme i e preiiveess aima mes r se iisver resear a i preiia sies sie e iiiai e dAs criia Pa


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tABLeS

Table 1.1. Major Uses o Animal Models and Other Model Organisms ................................................. 2

Table 2.1. Neuroprotective Genes Identied in C. elegans Parkinsons Disease Model .......................22

Table 2.2. Other Putative Neuroprotective Genes in Parkinsons Disease as Demonstrated inNon-C. elegans Systems ........................................................................................................................23

Table 4.1. Selected Genes Involved In Development o the Melanocyte Lineage and TheirPotential Roles in Melanoma ................................................................................................................50

Table 6.1. Comprehensive Strategies to Improve Mouse Models ......................................................... 77

Iiiaive i 2004? ................................................................................................................139

Qesi 10. d expe a imprvemes i e preiiveess aima mes r se

i isver resear a i preiia sies i e ex fve ears? ...........................139

Qesi 11. d expe ma ear mes ase ie pripe sem esr simiar eg repae sme ses aimas i parmaeia/iegresear ver e ex fve ears? .......................................................................................140

Qesi 12. des r mpa se meig/simai mve rm aima sies iisver a preiia sies i ma rias?..........................................................140

Qesi 13. d expe mper mes (vira aima mes, vira mames, vira psigia ssems, vira mrs, c.) repae sme ses aima mes ver e ex fve ears?..............................................................................140

Qesi 14. Is evepme mper-ase aima r ma mes severe imie researers imie kwege igia ssems a isease ig? .................141

Qesi 15. hw regais esige prme aima weare (.., e AimaWeare A, e Pi hea Servies Gie r e care a use lararAimas, a regais, e 3Rs) ae r perais? ...........................................141

Qesi 16. des r mpa wrk wi a e wig evep ve aimames? .................................................................................................................................141

referenCeS.....................................................................................................................................143

CompAny Index wIth weB AddreSSeS.........................................................................157


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Table of Contents

fIGUreS

Figure 3.1. The Role o Centrosomes in Mitosis ...................................................................................34

Figure 3.2. The TORC1 and TORC2 Pathways (Simplied Schematic) ............................................ 39

Figure 4.1. The Ras Pathway and Role o BRAF (Simplied Diagram) ............................................... 52

Figure 7.1. Zinc-Finger Nuclease-Mediated Gene Disruption ..............................................................91


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ntroduction

The adoption o invertebrate and zebrafsh animal models by industry has been driven bythe advent o genomics. Although genomics and proteomics have provided researchers with

a wealth o inormation about genes and proteins in humans, very little is known aboutthe unction o the vast majority o these genes and proteins, and their roles in normalphysiology and disease. Researchers use model organisms in many ways to attempt to gaininormation on the unctions and disease relevance o genes and proteins. This is possiblebecause not only genes, but also pathways, tend to be conserved during evolution.

1.1. Uses f mdel organiss

Major uses o animal models and other model organisms (e.g., bacteria and yeasts) aresummarized in Table 1.1.

table 1.1. majr Uses f Anial mdels and oher mdel organiss

Basic research

Biomedical research ( e.g., development o animal models o disease, understandingbiochemical and disease pathways)

Determining the cellular, genetic, and molecular basis o development

Studies o evolutionary biology and evo-devo

Developing new therapeutic strategies

Developing and utilizing novel animal models o human disease, in order to aidunderstanding of the disease and to develop and test experimental therapies

Overlaps with basic biomedical research utilizing animal models

Target evaluation

Gene, protein, and target discovery and characterization

Comparative genomics/proteomics, aimed at fnding related genes or proteins inhumans and model organisms via computational searches

Gene and protein expression proling

Biochemical pathway determination

Target validation (via gene knockout or gene knockdown with RNAi or antisensecompounds, and/or by overexpression of target genes)

Drug screening and lead optimizationEngineering small animal models ( e.g., C. elegans, Drosophila, and zebrafsh embryos)

or use in high-throughput screening

Preclinical studiesAbsorption, distribution, metabolism, excretion, and toxicity (ADMET)

Efcacy studies

source: Haberman Aociate


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Gee disrupio techoogies, fucioa Geomics, a targe Vaiaio

Since the development o knockout mouse technology by Mario Capecchi, Martin Evans,and Oliver Smithies in 1989 (resulting in their being awarded the Nobel Prize in Medicinein 2007), researchers have developed several tools or disrupting genes and thus determiningtheir unction. These include gene targeting via homologous recombination (the knockoutmouse technology), insertional mutagenesis using transposons, and gene trapping.104 Forexample, Lexicon Pharmaceuticals (The Woodlands, TX) developed a proprietary genetrapping method based on a retroviral vector that caused random insertional mutagenesiso genes in embryonic stem (ES) cells. Their technology enabled researchers to selectES cells with mutated genes and identiy sequence tags rom the genes. For unctionalstudies, Lexicon researchers used the selected mutant ES cell line to produce live mice, asin traditional knockout mouse technology. Enabled by automation, Lexicons gene trap

technology enabled it to industrialize the production of knockout mice. Lexicon was oncean animal model technology platform company known as Lexicon Genetics, but it morphedinto a drug developer that uses its gene trap technology as part o its drug discovery platorm.

Knockout mouse, gene trapping, and other gene disruption technologies have and continueto constitute a set o valuable tools or unctional genomics or basic researchers and indrug discovery. In particular, corporate researchers have used these technologies in targetvalidation. However, disease-related variants or mutations in humans are usually not causedby insertional mutagenesis. Moreover, one cannot use insertional mutagenesis to investigatemutations in regulatory and non-coding regions o DNA. It is thereore desirable to fnd

or produce mutations in mice that resemble the types o mutations involved in humandiseasepoint mutations (as in human SNPs) and copy number variants.

naura Variaio a Quaiaive trai loci

An important approach to fnding mutations the same as or similar to those ound inhuman traits and diseases is to perorm orward genetic studies on the many inbred mousestrains that have been produced by researchers over the years.104, 109 As with humans, manyo these mouse strains have variants that constitute quantitative trait loci (QTL), i.e.,loci that contribute to the variability of a complex quantitative trait such as body weight,blood pressure, or longevity. Inbred mouse strains (i.e., natural mouse strains that are not

transgenic or articially mutagenized) may also exhibit phenotypes, such as obesity, diabetes,hypertension, high incidence o leukemia, etc., which resemble human diseases. In mostcases, these phenotypes represent complex, multigenic (also called polygenic) traits. Asdiscussed earlier, complex human diseases also are multigenic and involve contributions bymultiple variants. Thus inbred mouse strains may be used to model certain complex humandiseases or traits (such as body weight) that are related to disease.

Forward genetics studies with inbred mouse strains in order to map QTLs are perormed bycrossing two inbred strains or several generations, in order to achieve segregation o QTLs.QTLs are placed on chromosome maps, which are defned by locations o such markers as

SNPs and short sequence length polymorphic (SSLP) markers.


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animals (or animals with gene duplications, etc.) via homologous recombination with thedonor DNA.

Sangamos zinc-nger nucleases (ZFNs) consist o zinc-nger domains linked to the DNAcleavage domain o the restriction endonuclease Fok1. Zinc-nger proteins (ZFPs) arenaturally-occurring, specic DNA-binding domains o transcription actors, each o whichconsists o several zinc ngers. Zinc ngers are small protein domains that utilize zinc ions tohelp stabilize their olds. Researchers can produce engineered zinc-nger proteins that bindto specic DNA sequences. This is the basis o Sangamos proprietary technology platormthat the company uses or various applications, including development o therapeutics and(in the case that is the ocus o this section) novel animal models.

The process o ZFN-mediated gene disruption is shown schematically in Figure 7.1.

figue 7.1. Zic-fige nuclease-meiate Gee disuptio

Target exon

Right ZFP

Fok1

Left ZFPZFN-induced cleavage

Repair by non-homologous end joining

Fok1

Zinc-nger nucleases (ZFNs) consist o zinc-nger proteins (ZFPs) (in this case consistingo ve zinc-nger moieties designed to bind to specic nucleotide sequences) linked toa cleavage domain o the restriction endonuclease Fok1. Each o the two ZFPs has beenengineered to bind to the right- or left-hand side of the desired cleavage site in a target exon.The ZFNs have been designed so as to require engagement o simultaneous binding o twoZFNs with a right- and let-hand ZFP domain, respectively, to give dimerization o the twoFok1 cleavage domains, resulting in double-strand cleavage o the DNA. The cell goes onto repair the break via non-homologous end joining, an imperect process that results indisruption of expression of the target exon.

source: Haberman Aociate


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Emerging Mammalian Model Systems

In order to validate their technology or producing knockout rats, the researchersconstructed three pairs o ZFNs that targeted the gene or green fuorescent protein (GFP)

and two endogenous rat genes. They administered each ZFN pair by microinjection intorat embryos; in the case o the ZFNs or GFP, they administered the ZFNs to a strain otransgenic rats carrying one copy o the gene or GFP in its germline. Screening o 295animals derived rom these embryos yielded 35 rats (12%) that had targeted mutations. TheZFN technology is thus a highly ecient method or producing gene disruption in rats. It isalso the fastest method, with a turnaround time of approximately four months.

In the case o the GFP rats, two o ve pups born ater microinjection o the GFP-targetingZFNs had no GFP expression and lost the green color seen in the parental strain. Sequencingo the targeted DNA regions o these animals showed that they had gene deletions in the

gene or GFP. Similar results were seen with rats in which endogenous rat genes had beentargeted. No gene disruptions were detected at any o the predicted o-target sites or theZFNs. This high rate o delity was made possible by the improved design o the ZFN pairs,which require dimerization o ZFNs carrying a right and a let hand-site (Figure 7.1) toachieve cleavage.142

Sigma-Aldrich Advanced Genetic Engineering (SAGE) is marketing knockout rat modelsbased on the ZFN technology (www.sageresearchmodels.com). SAGEs rat knockout modeldevelopment is ocused on the areas o basic (including drug discovery) and preclinicalresearch in the elds of neurobiology, cardiovascular disease, immunology, and toxicology.

The company has already developed several knockout rats and has more in development.SAGE also oers a custom knockout rat development service.

The ZFN disruption technology has also been applied to producing knockout zebrash andcould in principle be applied to mammals other than rodents.138

7.5. Ceatig Kockout mice a rats o Cultue SpeatogoialSte Cells

Spermatogonial stem cells (SSCs), isolated rom the adult testis, have the capacity bothor sel-renewal (i.e., prolieration to produce more o themselves) and or dierentiationinto spermatozoa. They thus orm the basis or producing gene-modied animals, includingin mammalian species or which ES cell derivation is technically dicult.138 This strategyinvolves isolating and culturing SSCs, perorming genetic modication o these cells, andtranserring the gene-modied SSCs to the germ cell-depleted testes o male animals. Thesemales are then mated with wild-type emales to yield heterozygous animals. Specic genetargeting can be done, for example, by using viral vectors based on rAAV, recombinantlentiviruses, and linear DNA (the latter similar to that done with ES cells in creatingknockout mice), in order to produce gene-modied (knockout or transgenic) animals.Random gene disruption, as described later in this section, may also be used to create

knockout mammalian models.


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8.4. Conclusions

Pharmaceutical and biotechnology researchers have been increasingly applying PD/PKmodeling, especially mechanistic PD/PK modeling, to all stages o drug development. Thisespecially includes moving rom preclinical animal studies to human clinical trials. Thesemodels are the most widespread and important computer-based mathematical models usedin drug development today. In the transition rom animal models to human studies, animportant ocus is dealing with dierences between animal models and humans, not onlywith respect to size (e.g., allometric scaling) but also with respect to other characteristicssuch as dierences in metabolism and the heterogeneity o the human population. Othertypes o mathematical modeling, such as Novartis biophysical modeling o the spinalcolumn, may also be employed to deal with specifc dierences between animal models andhumans that may be relevant to clinical trials o particular types o drugs (in this case, a

monoclonal antibody drug to treat spinal cord injury).

Entelos virtual patient models are being widely applied across the pharmaceutical industryas adjuncts to wet-lab drug discovery studies, animal studies, and clinical trialsin order toimprove the time and cost actors in drug development and the success rate o clinical trials.One o their uses is in the design o early clinical trials. Thus, or companies that use thesemodels, they (in addition to PD/PK models) are involved in moving rom preclinical toclinical studies. However, they are human physiology models, not virtual animal models.Moreover, they do not model deep molecular, biochemical, and cellular mechanisms odisease biology or drug action. Thus, especially or development o novel therapeutic

strategies and innovative drugs, they are at best adjuncts to wet-lab biology and animalmodel studies.

There is one model developed by Entelos that can be described as a virtual animal model.That is the virtual NOD mouse. This mathematical model is made possible by the extensivestudies that have been carried out or over 30 years with the living NOD mouse and thediabetes research communitys acceptance o its useulness and relatively aithul modeling ohuman disease. For animal models that have proven to be poorly predictive o human disease(e.g., most preclinical cancer and CNS disease models, which are the extreme examples),creating such a virtual model would not be possible. The same is true or novel animal

models that lack the extensive characterization of the NOD mouse.

Even in the case o the virtual NOD mouse, the model is an adjunct to real animal studies,not a substitute. However, it may enable researchers to use ewer animals and conductmore eective animal studies and hopeully more eective and successul clinical trials.Nevertheless, the useulness o the virtual NOD mouse in enabling researchers to discoverinnovative drugs that achieve proo o concept in clinical trials, let alone reach the market,remains to be confrmed.

The general limitations o computer models or creating useul virtual animal models that

can replace real animal models, discussed at the beginning o this chapter, remain valid.This is especially true given the increasing numbers o unknowns in the normal and disease


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Outlook

that are covered by these regulations in research, some studies (not only disease pathwayresearch and lead identication but also safety and toxicity screening) may be done in

invertebrates and zebrafsh embryos, provided that such usages are validated in theseorganisms. Even though the later stages o drug discovery and preclinical studies will be donein mammalian models, the numbers o mammals used will be reduced as required by theregulations.

9.4. Aial mol Stuis hl rsacs La About nw Asctso disas Biology

As we have discussed throughout this report, there are numerous aspects o normal anddisease biology that have only come to light very recently. These include such areas as the

biology o small regulatory RNAs (e.g., RNAi, microRNAs, snoRNAs), the importanceo copy number variation and non-coding regions o DNA in determination o diseasephenotypes, the importance o epigenetic regulation, and the biology o stem cells andits implications or normal and aberrant cell dierentiation. Moreover, GWAS in humangenetics have shown that common variants account or only a small raction o theheritability of complex diseases, indicating that researchers have a lot to learn about diseasebiology. In addition to these global issues in normal and disease biology, there are alsomore specic areas of disease biology that have recently come to light. Three examples ofsuch specifc areas that are discussed in this report are the importance o the epithelial tomesenchymal transition in cancer biology, mechanisms o genomic instability that can lead

to cancer, and the role o the biology o cilia in normal development and in various diseases.

Animal model studies, including studies that involve the creation o new disease models,have helped researchers learn about these areas o normal and disease biology, and willcontinue to do so. We have given some specic examples in this report. Both studies inmammalian systems as well as in invertebrates and fsh models contribute to this learningand its application to novel therapeutic strategies or human disease.

9.5. dvloig mo pictiv Aial mols o dug efcacy

We discussed the issue o developing more predictive mouse models o drug efcacy inChapter 6. (Since the ocus on predictivity o animal models is in the late stages o drugdiscovery and the preclinical stage, the ocus in this report has been on more predictivemouse models.) The two main reasons or researchers difculties in producing predictivemouse models are 1) dierences between the mouse and humans, and 2) major unknownactors in disease biology, as discussed in the previous section. Researchers can use animalmodels to learn about unknown or poorly understood areas o disease biology and in theprocess develop improved animal models, using the comprehensive approaches to developingimproved models discussed in Chapter 6 and the more specifc approaches (e.g., novel cancermodels based on the biology o tumorigenesis in humans) discussed in the case studies in the

same chapter. These specifc approaches oten involve applying knowledge o human diseasebiology to the creation o a new animal disease model. Researchers can also bridge some othe dierences between the mouse and humans by creating humanized mouse models


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nsight Pharma Reports Animal Models Survey: January 2010

Question 11. do you expect human cellular moels base on inucepluripotent stem cells or similar technology to replace some uses o

animals in pharmaceutical/biotechnology research over the next fveyears?

Yes

No

The new cellular models are mainlycomplementary to animal models

Response %

37.2%

16.7%

46.2%

n = 77

souce: Inight Phama repot

Question 12. does your company use moeling/simulation to moverom animal stuies in iscovery an preclinical stuies into humantrials?

Yes

No

Dont know

Response %

41.0%

47.4%

11.5%

n = 77


Question 13. do you expect computer moels (virtual animalmoels, virtual human moels, virtual physiological systems,virtual tumors, etc.) to replace some uses o animal moels over the

next fve years?

Yes

No

Computer models are mainlycomplementary to animal models

Response %

16.7%

35.9%

47.4%

n = 77



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Compny Index ith web

addressesA

Actelion (www.actelion.com) ................................................................................................. 50

AstraZeneca (www.astrazeneca.com) .......................................................................... 41, 104

Aveo Pharmaceuticals (www.aveopharma.com) .................................................................. 73

B

Bayer (www.bayer.com) .......................................................................................... 26, 48, 104

Bayer Schering Pharma (www.bayerscheringpharma.de/scripts/pages/en/inde).............26

Bayer-Schering ..................................................................................................................... 8, 57

Bristol-Myers Squibb (www.bms.com) ......................................................................... 25, 104

C

Cequent Pharmaceuticals (www.cequentpharma.com)....................................................... 85Charles River (www.criver.com)................................................................55, 87, 93, 121-124

Cytokinetics (www.cytokinetics.com)................................................................................. 25

d

DanioLabs ...........................................................................................................................56-57

deCODE genetics (www.decode.com)

.............................................................................................................................................. 7, 72


25/33

Company Index

e

Elixir Pharmaceuticals (www.elixirpharm.com)........................................................................ 5Entelos (www.entelos.com) ............................................................................103-105, 107, 155

Evotec (www.evotecoai.com) ...................................................................... 8, 56-57, 115-119

Exelixis (www.exelixis.com) ................................................................................................. 55

G

Genentech (www.gene.com) .......................................................................................... 40, 74

genOway (www.genOway.com) ............................................................................................. 1

GlaxoSmithKline (www.gsk.com) .................................................................................. 5, 102

I

Infnity Pharmaceuticals (www.inf.com).........................................................................82-83

iPierian (www.izumibio.com)....................................................................................... 10, 145

J

The Jackson Laboratory ................................................................1, 55, 65, 70, 75, 85, 124-126

Johnson & Johnson (www.jnj.com) .......................................................... 8, 57, 104, 118, 153

L

Lexicon Pharmaceuticals (www.lexpharma.com) .................................................... 55, 69, 75

Lilly (www.lilly.com)............................................................................................. 82, 102, 104

m

Maccine (www.maccine.com) .............................................................................................. 17

Merck (www.merck.com) ..................................................................7-8, 57, 72, 82, 104, 129

Merck KGaA (www.merck.de)......................................................................................... 8, 57

Merz (www.merz.com) ...................................................................................................... 8, 57


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n

Novartis (www.chiron.com) ............................................ 7, 14, 48, 55, 68, 102, 104, 107, 155Novartis Institutes or BioMedical Research (nibr.novartis.com) ........................................ 55

o

Open Monoclonal Technology ................................................................................................ 90

OSI (www.osip.com) ............................................................................................................. 41

p

Pfzer (www.pfzer.com) .................................................................................. 14, 26, 102, 104

Pharsight (www.pharsight.com)......................................................................................... 102

Phylonix (www.phylonix.com) ....................................................................................9, 55-56

Plexxikon (www.plexxikon.com) ......................................................................................... 51

r

Roche (www.roche.com)............................................................................... 8, 26, 51, 57, 104Rosetta Inpharmatics (www.rii.com) ......................................................................................... 7

S

Sangamo BioSciences (www.sangamo.com) ........................................................................ 90

sanof-aventis ........................................................................................................................... 25

Scynexis (www.scynexis.com) .....................................................................................130-131

Servier (www.servier.com)................................................................................................ 8, 57

Sigma-Aldrich Advanced Genetic Engineering ..................................................................... 92

Sigma-Aldrich Corporation..................................................................................................... 90

Simulations Plus (www.simulations-plus.com)................................................................. 102

Sirtris (www.sirtrispharma.com)...................................................................................... 5, 24

Summit Corporation ................................................................................................................ 57

t

Transposagen Biopharmaceuticals ........................................................................................... 93


27/33

Company Index

U

Union Biometrica (www.unionbio.com) ........................................................................ 29, 43

V

VASTox (www.wyeth.com) .................................................................................................. 57

w

Wyeth ....................................................................................................................................... 39

Z

Znomics (www.znomics.com) .............................................................................................. 56


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