RISC and Reward with Inhibitory RNAs

Information flow is fundamental to all biological systems, an attribute that helps us understand the nature and function of a cell, an organ or an organism. A central dogma of that flow is that it’s directional: The genetic code is deciphered from DNA to RNA intermediates, and finally to the structural and metabolic proteins from which we’re built. But a flaw in this orderly arrangement became clear by the end of the 20th century, as we began to understand that those RNA intermediates carry the informational flow in both directions, as messenger RNA translated into protein and as regulators of gene expression in the form of microRNAs and interfering RNA.Elbashir SM, Harborth J, Lendeckel W, et al. Duplexes of 21-nucleotide R11NAs mediate RNA interference in cultured mammalian cells. Nature 2001;411:6836:494-8. This month, we’ll look at the timeline of RNAi development and consider what the future may hold for this singular therapeutic modality.

ABCs of RNAs

Two Decades of RNAi

The process of RNA interference was comprehensively described by Andrew Fire, PhD, and Craig Mello, PhD,Fire A, Xu S, Montgomery MK, et al. Potent and specific genetic interference by double-stranded RNA in C. elegans. Nature 1998;391:806-811. in the late 1990s, and the signifi cance of this discovery was instantly recognized. Drs. Fire and Mello were awarded the 2006 Nobel Prize in medicine, and before that prize was even announced, Sirna Therapeutics had initiated a Phase I clinical trial for treatment of age-related macular degeneration with an interfering RNA targeting the type 1 vascular endothelial growth factor receptor.https://clinicaltrials.gov/ct2/results?term=NCT00363714 accessed 30 June 2015. While that trial yielded encouraging data, it didn’t progress.Kaiser PK, Symons RC, Shah SM, et al. RNAi-based treatment for neovascular age-related macular degeneration by Sirna-027. Am J Ophthalmol 2010;150:33-39. Despite this, the potential for this treatment approach was validated and efforts to develop small interfering RNAs as a therapeutic platform for a host of ophthalmic conditions have accelerated over the past decade.

Theoretically, a targeted siRNA has the potential to provide treatment for any human disease by interfering with disease-associated genes in a sequence- specific manner.Thakur A, Fitzpatrick S, Zaman A, et al. Strategies for ocular siRNA delivery: Potential and limitations of non-viral nanocarriers. J Biol Eng 2012;6:1:7. Designing small RNA fragments to interfere with mRNA in the cytoplasm has the added theoretical benefit of inhibiting the downstream synthesis of targeted proteins.6 This new therapeutic magic bullet was of particular interest for targeting genes that had traditionally been considered “undruggable” by other methods, such as small molecules. Moreno-Montanes J, Sadaba B, Ruz V, et al. Phase I clinical trial of SYL040012, a small interfering RNA targeting β-adrenergic receptor 2, for lowering intraocular pressure. Mol Ther 2014; 22:1:226-32. , Kanasty R, Dorkin JR, Vegas A, et al. Delivery materials for siRNA therapeutics. Nat Mater 2013;12:11:967-77.

The discovery of RNAi as a potential therapeutic modality led to an era filled with bidding wars for RNAi intellectual property and publication of a number of controversial findings in high-profile journals. Early efforts to exploit RNAi technologies did not live up to their hype, and thus a period of general backlash and financial restrictions ensued. Haussecker D. The business of RNAi therapeutics in 2012. Mol Ther Nucleic Acids. 2012;1:2:e8. After great initial promise came a series of disappointments followed by a steady progress to therapeutic success. Many of the therapeutics in the clinical pipeline now benefit from the scientific rationale derived from the historical trial-anderror of earlier RNAi therapeutic efforts, so we may have reached a second phase of therapeutic progress, with a growing number of RNAi therapies currently in various phases of clinical trials. Despite the mothballing of Big Pharma RNAi programs in the past, it seems that it is more of a question of when, rather than whether, RNAi therapeutics will reach their potential as a unique and valued treatment modality.

RNA interferenceDouble-stranded RNAs (dsRNAs) are processed into siRNA duplexes by the enzyme DICER. These short, dsRNAs are subsequently unwound and assembled into the RISC, which can direct RNA cleavage and translational repression using antisense strands from either endogenous or exogenous dsRNAs.


The endogenous process of transcribing genetic signals from DNA to RNA, and ultimately expressing proteins, is an evolutionarily conserved, multistep pathway that involves tissue- specific microRNAs and a series of enzyme complexes that drive the regulatory process. The miRNAs are trimmed to size by a specific ribonuclease called DICER and then paired with a target mRNA in a multiprotein RNA-induced silencing complex called RISC. The complexity of this cellular apparatus serves to underscore the importance of miRNAs as regulatory molecules. In the eye, miRNAs are increasingly used as biomarkers of disease states, and have been specifically linked to a host of ocular conditions. In the cornea, recent reports identify specific miRNAs that may participate in cellular events of wound healing. An J, Chen X, Chen W, et al. MicroRNA expression profile and the role of miR-204 in corneal wound healing. Invest Ophthalmol Vis Sci 2015;56:3673–3683. , Funari VA, Winkler M, Brown J, et al. Differentially expressed wound healing-related microRNAs in the human diabetic cornea. PLoS One 2013 Dec 20;8:12:e84425. By examining up- and downregulation of miRNAs associated with specific states, it’s been possible to identify new mechanisms underlying both healthy and pathologic healing processes.

It’s at this point in the endogenous gene regulatory process that therapeutic siRNAs enter. Whether generated by DICER or introduced exogenously, short, 18- to 25-nucleotide RNA duplexes assemble into the RISC and the double-stranded siRNA is separated, resulting in a single RNA strand coupled to RISC. The resulting RISC/ RNA strand complex identifies and cleaves complementary host mRNA, thereby preventing translation and selectively silencing gene expression. Although longer, double-stranded RNA has the potential to be delivered therapeutically, it is generally accepted that siRNA technology offers the best combination of specificity, potency and versatility as a therapeutic.De Fougerolles A, Vornlocher HP, Maraganore J, et al. Interfering with disease: A progress report on siRNA-based therapeutics. Nat Rev Drug Discov 2007;6:6:443-53.

Since siRNA must reach the cytosol of the cell to trigger RNAi, chemical modifications are required to bring siRNA to its site of action without inducing adverse effects, and to minimize recognition by the innate immune system. The potential for RNAi formulations to activate innate immunity was a significant hurdle for the early efforts to develop RNAi therapies. This issue has been largely circumvented by a combination of chemical modifications to the introduced RNA and by alterations in RNA structural design. In addition, newer siRNAs incorporate principles that ensure proper strand selection, avoid partial hybridization to non-target mRNAs, and thus minimize potential off-target gene silencing.

SiRNA and Ocular Disease

Early efforts to apply siRNA therapeutics to ocular conditions targeted retinal degenerative diseases due to the number of conditions with limited successful therapeutic options and difficulty in formulating topical application of siRNA therapy that will be stable and penetrate the ocular surface. These studies faced the same difficulties that all retinal drugs do: physical barriers; rapid clearance; and heterogeneous disease etiology. The isolated compartment of the eye, however, provides advantages for siRNA delivery compared to other tissues and organ systems in that the siRNA can be directly delivered. Lee DU, Huang W, Rittenhouse KD, et al. Retina expression and cross-species validation of gene silencing by PF-655, a small interfering RNA against RTP801 for the treatment of ocular disease. J Ocul Pharmacol Ther 2012;28:3:222-30. While most siRNA programs to date have delivered therapeutics by direct intravitreal injection,5 opportunities for new delivery methods are likely to impact the future success of siRNA in the eye.

The first clinical application of RNAi-based therapy was in 2006 by the intravitreal injection of a siRNA, Cand5, targeting vascular endothelial growth factor for the treatment of AMD. Although Cand5 showed initial promise in clinical trials, it was terminated in Phase III for its lack of efficacy. Ahmed Z, Kalinshki H, Berry M, et al. Ocular neuroprotection by siRNA targeting caspase-2. Cell Death Dis 2011;16;2:e173. By 2011, two additional siRNAs had been evaluated by direct intravitreal administration for their treatment of retinal degenerative diseases in humans. Sirna-027 [sic] was also designed to target the VEGF pathway by silencing the VEGF receptor. Like Cand5, Sirna-027 was studied in trials for the treatment of AMD but was terminated in Phase II. An additional siRNA, PF-655, also developed for the treatment of AMD, was made to silence RTP801, a propriety gene target owned by Quark Pharmaceuticals.7

Silencing the synthesis of the apoptotic protein, caspase-2 is another target for siRNA-mediated ocular therapeutics. In animal models, QPI-1007 has been shown to provide ocular neuroprotection through preservation of retinal ganglion cells. QPI-1007 has successfully completed Phase I studies for the treatment of non-arteritic ischemic optic neuropathy, and is also being evaluated in Phase II for acute, primary angle-closure glaucoma.

Another avenue for the application of siRNA for ocular disease therapy comes from Sylentis. The siRNA SYL040012 was developed as a therapy for primary open-angle glaucoma; it’s designed to inhibit the synthesis of the β2-adrenergic receptor, ADRB2. In contrast to previous siRNA therapies that are delivered by intravitreal injection, SYL040012 is the fi rst RNAi therapeutic to be administered as a topical formulation. SYL040012 is currently being assessed in a Phase IIb clinical trial for POAG.

The newest siRNA to enter clinical development, SYL1001, is also from Sylentis. SYL1001 targets the gene TRPV1 that encodes the receptor for capsaicin, the active ingredient in chili peppers. The gene product of TRPV1 is a key component of nociceptive sensory nerve endings, and silencing of this receptor via siRNA will be evaluated for the treatment of ocular pain associated with dry-eye syndrome in a Phase Ib study.

The Future of siRNA Therapies

Despite the $2.5 to $3 billion that was invested in RNAi therapeutics from 2005 to 2008, we have yet to see such a therapy come to market. A huge barrier to successful siRNA therapeutics has been their intracellular delivery, which significantly impacts their clinical efficacy. siRNAs are large and negatively charged, so they must be chemically modified or formulated to promote tissue distribution and cellular uptake. It is likely that future siRNA therapeutics will be designed with new drug delivery systems that will have a profound impact on improving the therapeutic outcomes for ophthalmic applications. One group of researchers has recently predicted that nanocarriers may be this delivery system, due to their reported ability to increase drug bioavailability while reducing side-effects and the need for repeated intraocular injections. Until then, there remains hope that products in development can demonstrate appropriate safety and efficacy. Despite its early promise, developing therapeutics that employ the siRNA platform has resembled swimming upstream, but recent success may indicate that going against the flow may ultimately be worth the risk.

Dr. Abelson is a clinical professor of ophthalmology at Harvard Medical School. Dr. Slocum is a medical writer at Ora Inc.