David R. Corey is in the Departments of Pharmacology and Biochemistry, Universityof Texas Southwestern Medical Center atDallas, 5323 Harry Hines Blvd., Dallas,Texas 75390-9041, USA.e-mail: david.corey@utsouthwestern.edu

RNA learns from antisenseDavid R Corey

RNA interference provides powerful tools for controlling gene expression in cultured cells. Whether RNAi willprovide similarly powerful drugs is unknown. Lessons from development of antisense oligonucleotide drugs may provide some clues.

Few discoveries have had the immediate and widespread impact of RNA interference (RNAi)1,2. The demonstration that short syn-thetic RNAs can inhibit gene expression in cul-tured mammalian cells unleashed a tidal wave of experimentation that has affected virtually every field of biological science. This tremen-dous success has created high expectations for developing RNA for treatment of disease. If RNAi works so well in cultured cells, can it be equally successful in people and create a revolutionary new class of therapeutics?

RNAi has made an impact because it pro-vides a natural mechanism for increasing the potency of synthetic nucleic acids. Cellular machinery—the RNA-induced silencing com-plex (RISC)3—recognizes the RNA duplex, facilitates pairing of one strand with its target mRNA (Fig. 1a), and prevents translation by destroying the target. By using RNA oligonu-cleotides, drug designers have the opportunity to work with nature rather than against it.

Duplex RNA has just entered phase 1 clini-cal trials (Table 1), and it will take years for its potential as a therapeutic modality to be eval-uated with confidence. It is not too early, how-ever, to analyze the challenges that will likely be faced during development and anticipate the areas that will require intense research. A logical starting point for this analysis lies with the older cousin of small interfering RNA (siRNA): antisense oligonucleotides.

Like siRNA, antisense oligonucleotides bind to mRNA4. However, unlike siRNA there seems to be no evolved cellular mechanism

for promoting antisense strand recognition, and it is likely that antisense oligomers must find their targets unassisted. Once bound, antisense oligomers can block expression via a “steric” mechanism by obstructing the ribo-some (Fig. 1b)5. Alternatively, antisense oligo-mers can form an RNA-DNA hybrid that can be a substrate for RNase H, thereby causing the target mRNA to be destroyed (Fig. 1c)6. Several of the most advanced antisense oligo-mers are “gapmers” consisting of a central DNA portion that can recruit RNase H and flanking 2´-modified regions that enhance stability to nuclease digestion and increase affinity to target sequences7. For clinical use, antisense oligomers (steric or gapmer) have phosphorothioate8 or morpholino9 backbone modifications to further increase resistance to degradation and to improve biodistribution.

We’ve been here beforeAntisense oligonucleotides have been vigor-ously pursued as a therapeutic strategy for over 20 years. One antisense oligonucleotide, fomivirsen, has been approved for treatment of cytomegalovirus (CMV) retinitis. US Food and Drug Administration (FDA) approval was a landmark for the field, but relatively few people have been treated; fomivirsen is administered by intraocular injection, mak-ing it a highly specialized case. Balanced against this success, three other oligonucle-otides that are administered systemically have failed to gain FDA approval after phase 3 clinical trials.

Why has progress been so slow? It is essen-tial to realize that the leading industrial and academic laboratories had to invent an entirely new field for drug development. Before their heroic efforts there was little information to guide to the development of large (molecular weight > 6,000) polyanionic drug candidates intended to work inside cells. It is hard to

imagine a class of synthetic compounds more dissimilar from traditional drugs. Pioneering work was necessary to (i) define mechanisms of action for antisense oligonucleotides, (ii) invent chemical modifications to improve stability, specificity, affinity, potency and bio-distribution, (iii) scale up syntheses so that systemic administration could be performed at reasonable cost, (iv) characterize pharma-cology and toxicology, and (v) develop an understanding of what constitutes a good tar-get gene in vivo. In retrospect it is not surpris-ing that success was not achieved as quickly as some might have hoped.

The failed phase 3 trials all used phos-phorothioate DNA oligonucleotides that had begun development in the early 1990s. Researchers at both company and academic laboratories were not content to wait for the results of these trials; instead they have pushed to develop second-generation com-pounds that take advantage of improved chemical modifications and a better under-standing of what genes can be good targets for drug development.

Early indications are that these efforts may be rewarded. Data from multiple phase 1 and phase 2 clinical trials (Table 1) are promising, which suggests that the combination of judi-ciously chosen targets and optimized chemical design can produce promising clinical candi-dates. For example, ISIS 113715, a chimeric gapmer oligonucleotide designed to target protein tyrosine phosphatase 1B (PTP1B) for treatment of type 2 diabetes, yielded a statistically significant improvement in glu-cose control in a phase 2 clinical trial. ISIS 301012, a gapmer targeting apolipoprotein B (apoB1), reduced concentrations of apoB1 in people and yielded a promising clinical profile. An important feature of the apoB1 study is the fact that the ability to monitor antisense efficacy by a simple blood draw

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provided convincing evidence of target-spe-cific knockdown in the clinic.

Lessons learned: a fast start for siRNA Previous experience with antisense oligo-nucleotides is directly relevant to the clinical progress of siRNAs. Indeed, siRNAs and anti-sense oligonucleotides have more similarities (Box 1) than differences. Just like antisense oligonucleotides, the vast majority of char-acterized siRNAs target mRNA. Scale-up

synthesis protocols will be similar, and the chemical modifications that have helped renew the promise of antisense oligonucle-otides are available to tailor the properties of siRNAs. Both duplex RNA and antisense oligonucleotides tend to localize to the liver upon systemic administration10,11, which makes the liver a prime organ to search for candidate target genes. Indeed, one of the most important lessons from antisense strate-gies is the need to understand oligonucleotide

biodistribution and focus on diseases that affect organs and cell types in which syn-thetic nucleic acids accumulate, because these are the organs in which nucleic acids may be most likely to have an impact after systemic administration.

The similarities between siRNA and antisense oligonucle-

otides have greatly benefited the preclinical and clinical development of duplex RNA. All of the hard-won experience of the antisense field was available at the inception of work with duplex RNA. For example, duplex RNA is much more stable than one might imagine12 (this is one reason for its enormous success as a laboratory tool), but it is not sufficiently sta-ble in vivo. This deficiency, however, was read-ily corrected by standard medicinal chemistry approaches using the rich toolbox of modi-fications originally developed for antisense oligonucleotides12–15. Likewise, early animal testing of siRNAs has been done in systems that closely mimic early work with antisense oligonucleotides16–19. It has been possible to use well-established antisense oligonucle-otides as positive controls, thereby eliminat-ing many of the variables that would have otherwise slowed development by obscuring interpretation of results. Because of this head start, progress that required 10–15 years for antisense oligonucleotides has required just 2–4 years for siRNA.

There are also significant differences between antisense oligonucleotides and siRNAs.

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Figure 1 Mechanisms for antisense oligonucleotides and siRNA. (a) Cleavage of mRNA mediated through RISC. A duplex RNA is recognized by the RISC complex. The complex facilitates recognition of the complementary mRNA sequence and then cleaves the sequence. (b) Steric mechanism for inhibiting gene expression. An antisense oligomer binds mRNA and blocks function of the ribosome or other proteins that are critical for elongation or splicing. (c) RNase H–mediated cleavage of mRNA. A gapmer oligonucleotide that contains a central DNA portion binds mRNA. The DNA-RNA hybrid is recognized by RNase H, which cleaves the mRNA. Adapted with permission from ref. 4, copyright 2002, American Chemical Society.

BOX 1 SIMILARITIES BETWEEN ANTISENSE OLIGONUCLEOTIDES AND siRNASShort (~20 base) nucleic acidsSynthesized by similar methodsSystemic administration results in primary localization to liverLocal administration is an option for many diseasesComplementary to mRNACan induce target RNA destructionCan incorporate chemically modified basesChemical modifications necessary for useful in vivo propertiesOngoing testing in multiple clinical trials

From ref. 24.

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siRNAs have two strands, and recognition is mediated through the RISC complex (Fig. 1a). Antisense oligonucleotides have one strand that can block RNA by a steric mechanism (Fig. 1b) or form a DNA-mRNA hybrid that recruits RNase H (Fig. 1c). Because of these differences the antisense experience will not be a perfect predictor for all of the challenges faced by duplex RNAs.

Antisense or siRNA? Neither? Both?The similarities between siRNAs and anti-sense oligonucleotides raise an important question: will siRNAs be superior in vivo? The popularity of siRNA suggests that the strat-egy has advantages for silencing expression in cultured cells, but will there be advantages in people? Initial animal studies have demon-strated that siRNA can reduce gene expres-sion, but there is no indication that silencing is substantially better than that achieved by antisense oligonucleotides. Antisense oligo-nucleotides, moreover, have three inherent advantages: (i) they are half the molecular weight, (ii) they do not require a hybridiza-tion step, thus simplifying large-scale prepara-tion of drugs, and (iii) because endogenous duplex RNAs control important physiologic processes, introducing a synthetic RNA may perturb this native machinery. This last point has become a concern because recent studies have shown that duplex RNAs can compete for binding to RISC20 and cause toxicity in vivo21. These potential advantages for antisense are balanced against the enticing potencies shown by siRNAs in cultured cells. These contrast-ing strengths may have substantial effects on biodistribution, potency and toxicity.

It is tempting to speculate that antisense oligonucleotides may have chemical and bio-logical properties that will be superior for some target genes and tissues, whereas siRNAs may be superior for others. For example, in one tissue the uptake or the efficiency of the RISC complex may favor siRNA. In another tissue, single-stranded antisense oligomers may enter more efficiently. At this early stage of development, having multiple options for improving biological activity will likely prove important.

The theoretical discussion of the relative value of siRNA and antisense oligonucle-otides can only be resolved by clinical devel-opment. Once again, clinical plans for siRNA have benefited from the antisense experi-ence. Both Sirna Therapeutics and Acuity Pharmaceuticals are conducting trials that use siRNAs to target vascular endothelial growth factor (VEGF) to treat macular degeneration. These trials take advantage of the small, iso-lated volume within the cornea, the fact that

VEGF is a validated target for approved ther-apies (it has been targeted by pegaptanib22, an aptamer, and ranibizumab23, an antibody fragment developed by Genentech), and the Isis Pharmaceuticals experience of obtain-ing approval for a nucleic acid drug admin-istered by intraocular injection. Similarly, the lead compound ALN RSV01 at Alnylam Pharmaceuticals is designed to treat respira-tory syncytial virus (RSV), a target in the lung that may be susceptible to local delivery by inhalation.

Delivery by local administration is a good strategy for these first clinical trials because it avoids the biodistribution challenges of systemic delivery and reduces the need for compound. Local delivery also facilitated the progress of Isis’ approved antisense drug fomivirsen, and success with these siRNAs would be a case of history repeating itself.

The trials by Alnylam, Sirna and Acuity will determine whether locally administered siRNAs can be a useful therapy. They will not define the full potential of siRNA or address whether it will provide the foundation of a new class of systemically administered therapeu-tics. Further work will be needed to improve in vivo potency. This might be achieved by careful choice of optimal target genes—genes that are susceptible to silencing and in which achievable levels of silencing have useful clini-cal effects. Almost certainly, however, potency will involve advanced medicinal chemistry to improve both biodistribution to tissues and uptake by specific cell types, and to reduce

off-target effects and enhance the efficiency of silencing by those duplexes that ultimately reach target cells.

ForwardWhat is needed for progress? There are no easy answers. There is no evidence for the existence of a magic bullet that will suddenly allow siRNA to live up to its potential as treatment for a broad range of diseases. Yet one suspects that in the large realm of chemical space a magic bullet (or bullets) does exist. Perhaps one or more chemical modifications will be discovered that enhance tissue uptake. Clever delivery methods may be discovered that direct siRNAs to diseased tissues. New formulations may be found that deliver RNA to a wider range of organs and cancerous tissues.

What is certain is that this development process would be greatly enhanced by the existence of a standardized in vivo testing sys-tem that allows innovative chemical modifi-cations, delivery strategies, and formulations to be tested and compared. No one lab has the expertise or funding to accomplish these tasks, which suggests an important organizing role for a large funding organization or insti-tution. Having many different laboratories attack these substantial problems in isolation, as is now the case, may not be the best strategy for fully exploiting the power of RNAi for the benefit of individuals.

No one knows what the future holds for siRNA as therapy. It is impossible to predict how many people will have their lives improved

Table 1 Partial listing of current clinical trials for antisense oligonucleotides and siRNAs

Drug (manufacturer) Type of compound Indication (molecular target)

Compounds in phase 3 trials

Genesense (Genta) Phosphorothioate DNA Varied cancer (BCL2)

Compounds in phase 2 trials

Alicaforsen (Isis) Phosphorothioate DNA Ulcerative colitis (ICAM1)

ISIS 301012 Chimeric gapmer High cholesterol (apoB-100)

ISIS 113715 Chimeric gapmer Diabetes (PTP1B)

ATL1102 (ATL/Isis) Chimeric gapmer Multiple sclerosis (VLA4)

OGX-011 (OncoGenex/Isis) Chimeric gapmer Prostate cancer (clusterin)

SPC2996 (Santaris) Locked nucleic acid B-cell lymphoma (BCL2)

Resten-NG (AVI) Morpholino Restenosis (MYC)

Compounds in phase 1 trials

LY21181308 (Lilly/Isis) Chimeric gapmer Cancer (survivin)

LY2275796 (Lilly/Isis) Chimeric gapmer Cancer (eIF4E)

GRN163L (Geron) Phosphothioamidate Cancer (telomerase)*

AVI-4065 (AVI) Morpholino Hepatitis C (HCV virus)

AVI-4557 (AVI) Morpholino Drug metabolism (cytochrome P450)

Cand5 (Acuity) siRNA Macular degeneration (VEGF)

Sirna-027 (Sirna) siRNA Macular degeneration (VEGF)

ALN-RSV01 (Alnylam) siRNA RSV infection (RSV virus)

*GRN163L targets the RNA component of telomerase rather than telomerase mRNA. All information was obtained from corporate websites.

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or extended, although it is a safe bet that this number will not be zero. What is predictable is that medicinal chemists, biologists and phar-macologists have learned from the antisense experience and are well prepared to undertake the methodical studies that will be necessary.

ACKNOWLEDGMENTSThe author has been a consultant for Isis Pharmaceuticals. The author acknowledges support from the US National Institutes of Health (NIGMS 60642 and 73042) and the Robert A. Welch Foundation (I-1244).

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