Does antisense make sense?

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4 1359-6446/99/$ see front matter Elsevier Science. All rights reserved. PII: S1359-6446(98)01282-3 DDT Vol. 4, No. 1 January 1999UPDATEUsing antisense oligonucleotides as highly selective pharmaceuticalagents to block expression of disease-associated proteins has been a tantaliz-ing promise for more than 20 years.Now that promise has become a reality:Isis Pharmaceuticals (Carlsbad, CA,USA) was granted an approval by theUS Food and Drug Administration(FDA) on 24 August 1998, to marketfomiversen sodium (Vitravene), anantisense oligonucleotide intended foruse in the treatment of AIDS patientswith cytomegalovirus (CMV)-inducedretinitis. Does this approval mark thebeginning of a new era in which theexquisite specificity of antisense tech-nology will be used to alter the courseof many different diseases, or is the ap-proval of Vitravene an anomaly unlikelyto be readily repeated?The concept behind antisense is sim-plicity itself: an oligonucleotide with se-quence complimentary to a region ofthe mRNA for a targeted protein is introduced into a cell. The oligonu-cleotide binds to that mRNA throughWatsonCrick base pairing to physicallyblock its translation and synthesis of itsspecified protein. An oligonucleotideonly 12 nucleotides in length has suffi-cient specificity to identify a particularmRNA, blocking, in theory, the synthe-sis of only the target protein encodedby that mRNA. Instead of being translated, the oligonucleotidemRNA complex is degraded by RNase H.Potentially, it is an elegantly specifictreatment for a host of diseases rangingfrom inflammation to cancer and frombacterial to viral infections, with little orno side effects.Tried and failedDespite the simplicity and promise ofthe concept, attempts to develop anti-sense drugs have met with many fail-ures, and many, if not most, drug dis-covery scientists have discounted theutility of the concept because of the dif-ficulties of getting the simple concept towork in practice. Because of these fail-ures, several companies involved in antisense technology, including GileadSciences (Foster City, CA, USA) andGenta (San Diego, CA, USA), havemoved their drug discovery efforts to other research areas. Hybridon(Cambridge, MA, USA) is still pursuingantisense programs with its own ver-sion of antisense treatment for CMV retinitis (Phase I) and research on anantisense drug for cancer. However,Hybridon had to cancel their programin HIV therapy, despite considerable fi-nancial investment, because of dose-related side effects. Even Isis had todrop its first antisense drug, a treatmentfor human papilloma virus, because thefrequency of treatment needed was notconsistent with a practical therapeutic.In spite of the nagging problems thathave plagued the use of antisense, IsisPharmaceuticals was built and con-tinues to be based upon the belief thatthe antisense technology will eventuallywork. Consequently, the approval ofVitravene was a particularly momentousevent in the life of the young company:not only is it the first drug with a claimfor an antisense mechanism to be ap-proved for marketing by the FDA; it isthe first product Isis has brought tomarket, and if it turns out to workthrough an antisense mechanism, it willprovide validation for the therapeuticstrategy upon which the entire com-pany is based.Special case?Vitravene is administered to patientssuffering from CMV infection by in-traviteral injection once weekly duringthe initial stages of treatment and thenevery two to four weeks during mainte-nance. The direct injection into the siteof infection circumvents many of theproblems associated with the systemicadministration of antisense therapeuticsand was quite probably one of themajor reasons that CMV infection waschosen as the first therapeutic target bythe young company. Despite this some-what special case for their first product,many other antisense drugs will followin its footsteps, claims Stanley T.Crooke, the Chairman of the Board andCEO of Isis, who left SmithKline &French to found the company in 1989.Stringent testingIsis has several other antisense productsin its pipeline that represent a morestringent test for the antisense strategy.In collaboration with BoehringerIngelheim Pharmaceuticals (Ingelheim,Germany), Isis is testing an antisensedrug for down-regulation of the adhe-sion protein ICAM-1 and its potentialfor use in the treatment of CrohnsDisease. This drug is currently in a piv-otal clinical trial. They also envisionthat ICAM-1 will be an effective anti-sense target for the treatment of psoria-sis (Phase II completed), rheumatoidarthritis, ulcerative colitis and tissue re-jection after transplantation (all in PhaseII). Other antisense drugs in variousstages of clinical trials target protein ki-nase Ca (Phase II), c-raf (Phase II), andHa-ras (Phase I) for the treatment ofcancer, and a second-generation anti-sense product is under investigation forthe treatment of CMV retinitis in AIDSpatients (Phase I).Research hurdlesThe major problems in developing anti-sense drugs have been due to the insta-bility of oligonucleotides in a world fullof nucleases, and difficulties with deliv-ery of the antisense reagents to the dis-ease site and into the target cell. [For areview of delivery strategies see Miller,K.J. and Das, S.K. (1998) Pharm. Sci.Tech. Today 1, 377386.] A consider-able amount of research also had to beDoes antisense make sense?UPDATEinvested in finding the right region oneach mRNA to hybridize with an anti-sense oligonucleotide. Originally it wasthought that hybridization of an anti-sense reagent to any portion of the tar-get mRNA would suffice to block trans-lation, but this reasoning was incorrect.Hybridization of an oligonucleotide tosome portions of the mRNA sequencehave no effect, while other regions pro-vide insufficient inhibition of translationfor the desired therapeutic response. Inpractice, it is necessary to exploredozens of different regions of the targetmRNA before a hybridization site isfound that allows the antisense oligonu-cleotide to shut down translation.Finally, considerable controversy hasarisen over the mechanism of action ofantisense drugs: most of the oligonu-cleotides used for the antisense strategybind nonspecifically to other moleculesin the cell and appropriate control ex-periments were not always performed,particularly in the early days of anti-sense research, which led to misleadingresults. Even when appropriate controlexperiments are conducted, determin-ing the specific mechanism of actioncan still be a challenge.Nuclease resistanceFinding a way to increase resistance tonuclease activity was one of the firstproblems to be dealt with successfully.Substitution of a sulphur atom for oneof the oxygen atoms in the phosphatebackbone of the oligonucleotide to pro-duce phosphorothioate oligonucleotideprovided a molecule with a marked in-crease in stability in the presence of nu-clease activity. Yet the modification stillallows the formation of the WatsonCrick base pairs necessary for hybridi-zation with a complimentary region of mRNA.There is a drawback, however, to the use of phosphorothioate oligonu-cleotides: they have a tendency to bindnonspecifically to proteins and RNA.Serum albumin, for example, bindsphosphorothioate oligonucleotides withan affinity in the range of 150 mM,which Crooke has noted is in a similarrange as the binding of aspirin or peni-cillin to serum albumin [Crooke, S.T. etal. (1996) J. Pharmacol. Exp. Ther. 277,923937]. Specific binding to proteins, depen-dent upon the nucleotide sequence orthe overall structure of the oligonu-cleotide, has also been observed andpresents a more serious problem for theinterpretation of antisense experiments.Control experiments usually use anoligonucleotide with a scrambled nu-cleotide sequence, which may not ex-hibit the same binding pattern to pro-teins as the therapeutic oligonucleotide,leading the investigator to conclude thatthe reagent is acting through an anti-sense mechanism, when in reality it isaltering the activity of a key protein.Because of these complications, a widerange of circumstantial evidence mustbe collected to support an antisensemechanism of action. Such evidenceincludes demonstrating the loss of target mRNA on Northern blots in a time-course consistent with thedoseresponse curve for the activity ofthe antisense reagent both at the celllevel and in the intact organism. Similarexperiments can be performed to in-vestigate the correlation of the rankorder potency of various antisenseagents and the loss of target mRNA andprotein both in vitro and in vivo.Persistent problemGetting the antisense drug to the dis-ease site is a problem that persists.Antisense reagents will probably alwaysbe limited to parenteral administration they are not readily absorbed intactwhen administered orally. Several stud-ies have shown, however, that upon in-jection by any of a variety of routes, an-tisense oligonucleotides have a half-lifeand distribution consistent with theiruse as therapeutic reagents. Finally,phosphorothioate oligonucleotides aretaken up and broadly distributed in awide variety of cell types in vitro, al-though the exact mechanism of uptakeis unknown.Remaining questionsIn spite of the advances in dealing withthe practical problems of antisenseoligonucleotides, the approval grantedto Isis by the FDA and the extensivedata collected by Isis on the mechanismof action of Vitravene, questions remainsurrounding the notion that Vitraveneproves the case for antisense therapeu-tics. The administration of Vitravene di-rectly into the eye does not test the gen-eral ability of an antisense reagent toreach a disease site upon systemic ad-ministration, and the possibility thatsome other unknown mechanism of ac-tion may account for the therapeutic ef-fect still cannot be conclusively ruledout. In fact, it is known that at highdoses, Vitravene has non-antisense ef-fects and the compound may actuallybind to the CMV coat proteins prevent-ing the cellular uptake of the virus.However, direct binding of Vitravene tothe coat proteins is not considered to bea mechanism of action of the drug, be-cause the virus is believed to be trans-mitted in the eye by direct cell-to-cellcontact without the release of free virus.There may be reservations, but Isisresults with Vitravene are exciting andhave succeeded in resurrecting the fieldof antisense therapeutics. If Vitravene isacting through an antisense mechanism,as Crooke asserts, and if some of theother antisense drugs in Isis pipelineare also successful in reaching the mar-ket, then the antisense strategy, despiteits many years of trials and tribulations,may yet pave the way for a new era ofhighly specific therapies for a widerange of diseases that currently have nospecific means of intervention.Robert W. Wallacetel/fax: 11 212 254 3322e-mail: RobWallace@nasw.orgDDT Vol. 4, No. 1 January 1999 5