siRNA: Set for a Comeback?

Although small-interfering RNA drugs faltered in recent trials, their potential merits continue to inspire research


siRNA: Set for a Comeback?

Although small-interfering RNA drugs faltered in recent trials, their potential merits continue to inspire research.


Ischemic vascular diseases of the choroid and retina are of particular epidemiological importance in causing visual impairment among the working and elderly populations. Neovascular age related macular degeneration is a leading cause of blindness over the age of 65 years. In the United States, neovascular AMD affects 1.75 million individuals over 40 years old.1

Vascular endothelial growth factor-A is important in both angiogenesis2 and vascular permeability.3,4 Increase in VEGF-A was found to be a key factor in both choroidal and retinal neovascularization. In recent years, treatments that inhibit VEGF became important tools for treating ischemic vascular disease of the retina. Accumulated data indicate that attenuation of VEGF activity could effectively suppress ocular neovascularization. Current treatments are based on antibodies against VEGF. However, although treatment-targeted antibodies against VEGF are effective, they are not efficient. The treatments require repeated administration due to transient effect, and large amounts of antibodies are needed to suppress the targeted protein. The injections are costly, and they increase the burden on both the patient, who is required to repeatedly attend the clinic, and on the physician. The repeated treatments also potentially increase the side effects associated with the injections.


RNA interference is a recently developed technique to silence proteins in a sequence-specific manner by inhibiting mRNA and consequently reducing protein expression. The functional mediator of RNA interference is a short double-strand RNA (dsRNA) oligonucleotide called small-interfering RNA (siRNA).1 siRNA, when present in the cytoplasm of the cell, is incorporated into a protein complex called the RNA-induced silencing complex (RISC). Argonaute 2, a protein within the RISC, unwinds the siRNA, after which the sense strand of the siRNA is cleaved. The activated RISC that contains the antisense of the siRNA degrades mRNA that is complementary to the antisense strand. The activated RISC complex can destroy additional mRNA targets (Figure). The therapeutic effect is dependent on the rate of the cell division and persists until the siRNA are diluted or degraded within the cell.5 Use of siRNA against isoforms of VEGF, such as Cand5, and siRNA targeted to VEGF receptors, such as siRNA-027, have been developed.6

Figure. Schematic of the steps leading to mRNA interference.

Rita Ehrlich is a vitreoretinal fellow at the University of Auckland, New Zealand. Thomas A. Ciulla is a partner at Midwest Eye Institute, Indianapolis. Neither author reports any financial interest in any product mentioned in this article. Dr. Ehrlich can be reached at


Small interfering RNA agents designed to silence the VEGF gene and the VEGF receptor have been shown in preclinical studies to inhibit ocular neovascularization and vascular permeability in animal models.7,8 Sirna-027 (also known as AGN 211745, Allergan Inc., Irvine, CA) is a chemically modified siRNA molecule that targets a conserved region of VEGF receptor 1 mRNA molecules. In a prospective, open-label, single-treatment, phase 1 study on patients with CNV due to AMD, a single intravitreal dose of Sirna-027 between 100 μg to 1,600 μg was well tolerated in patients who had been refractory to photodynamic therapy or photocoagulation treatment prior to the anti-VEGF injection era. Stabilization or improvement in visual acuity and foveal thickness was observed in most patients. On day 84, 14.4% of patients had improvement in visual acuity of three lines or more, and 7.7% had deterioration in visual acuity.9

A further phase 2 trial randomized patients between four treatment arms. The first arm consisted of three ranibizumab (Lucentis, Genentech) injections at monthly intervals. The three experimental arms consisted of different doses of the study medication to be administered three times at monthly intervals. The study failed to meet the efficacy endpoints.

Bevasiranib (previously called Cand5; Opko Health, Inc./Acuity Pharmaceuticals) is designed to block the production of VEGF directly by inhibiting the mRNA from the VEGF gene. In a phase 2 randomized trial, bevasiranib (single intravitreal injections at baseline and week 6) was shown to be well tolerated after 12 weeks of follow-up, with mild adverse events related primarily to the injection procedure and with no systemic exposure. Bevasiranib stabilized visual acuity in most patients and improved VA in more than one-third of patients.

The COBALT (COmbining Bevasiranib and Lucentis Therapy in wet AMD) was a phase 3 trial testing of this agent in combination with ranibizumab. The rational for the combination treatment is to block both production of new VEGF and existing VEGF by siRNA and ranibizumab, respectively. The phase 3 trial evaluated the safety and efficacy of bevasiranib given every eight or 12 weeks after ranibizumab pretreatment compared with ranibizumab alone every four weeks. More than 30% of subjects that received ranibizumab followed by bevasiranib had improvement of at least three lines of visual acuity. The greatest difference compared to the group treated with only ranibizumab was at six weeks.These preliminary results suggest a possible benefit of ranibizumab-bevasiranib combination therapy to treat AMD.10 However, Opko Health decided in early 2009 to terminate its phase 3 trial, after the Independent Data Monitoring Committee found that, although bevasiranib showed activity when used in conjunction with ranibizumab, the trial was unlikely to meet its primary endpoint.

Pfizer and Quark Pharmaceuticals' PF-4523655 siRNA is targeted to inhibit expression of the DNA damage-inducible transcript 4 (DDIT4) gene, also known as REDD1 and RTP-801. It is currently in phase 2 clinical trials in both neovascular AMD and diabetic macular edema. PF-4523655 inhibited expression of RTP801 in a dose-related manner, starting as early as the first day after its single intravitreal administration, for up to 14 days.

The MONET study is a phase 2 study comparing ranibizumab to combination treatments with ranibizumab and PF-4523655. The study arms include ranibizumab only, ranibizumab injection followed by 1 mg or 3 mg of PF-4523655 every two weeks from week 4 to week 12, 3 mg of PF-4523655 given every four weeks starting from week 4 to week 12, and combination treatment of ranibizumab and 1 mg PF-4523655 every four weeks starting from baseline to week 12.

The same agent is also being investigated in patients with DME in a study called DEGAS. The study investigates the efficacy of three different doses of intravitreal treatment compared to macular laser photocoagulation. Efficacy will be measured at 24 and 36 months in terms of visual acuity change, as well as changes in fluorescein angiographic leakage and optical coherence tomography measurements.

Still, the specificity of the aforementioned siRNA drugs was recently called into question by a recent study showing that the antiangiogenic activity of siRNA may be due to a class immune effect associated with activation of the cell surface toll-like receptor (TLR) 3, rather than to a target sequence-specific interaction. The authors further found that TLR induction was dependent on the length of the siRNA. DsRNAs of 21 base pairs or longer induced this response; smaller ones did not.11

Although this study questions the nature of the antiangiogenic effect of siRNA, it does not explain in full the therapeutic effects observed in some of the studies.5 Moreover, these siRNA drugs could theoretically cause vision loss via activation of the TRL3 receptor, which may promote the formation of geographic atrophy in genetically susceptible individuals.12 In future development of siRNA, attention will be required to potential nonspecific activation of the immune system by siRNA, including TLR3 and TLR7.


Li et al.13 proposed RNA interference as a gene silencing therapy to eliminate the mutant myocilin proteins in the trabecular meshwork cells in open angle glaucoma. Mutation in the MYOC gene that encodes myocillin has been linked in recent years to open angle glaucoma and is responsible for 3% to 4% of adult-onset open angle glaucoma cases. Mutation in the MYOC protein results in apoptosis in the trabecular meshwork cells, which can lead to increased resistance to aqueous humor outflow and raised intraocular pressure.

Robo1-specific siRNA was recently found to delay the onset of proliferative vitreoretinopathy in a rabbit model, although it was not shown to prevent it. Robo1 was found present in the extracellular matrix of proliferative membranes in proliferative vitreoretinopathy and proliferative diabetic retinopathy membranes. The Roundabout (Robo) is a family of proteins that are related to transmembrane receptors and play a major role in neurogenesis. Robo1 was found to be expressed in neural tissues, including nerve fiber layer, ganglion cell layer, inner and outer plexiform layers, and retinal vessels, during mouse retinal development.14

The mitogen-activated protein kinases (MAPKs) family consists of a large number of serine/threonine kinases that form a signal transduction system controlling cellular events, such as proliferation, differentiation, survival and apoptosis activated by oxidative stress.

siRNA-mediated gene silencing of S-phase kinase-interacting protein 2 (Skp2)15 and siRNA of nuclear factor kappa B (NF-kappaB)16 were found to inhibit migration and proliferation of lens epithelial cells and to decrease posterior capsule opacity formation in animal models. These experimental studies on siRNA-silencing treatment are intriguing, but the clinical implications of these treatments, if any, remain unanswered questions.


In the future, new dosing schedules, combination treatments and enhanced delivery systems might be considered. Other siRNAs targeted at various steps within the pathogenesis of AMD might be developed. These targets include VEGF-specific isoforms with VEGF-165B–sparing, hypoxia inducible factor-1 alpha (HIF-1 alpha) and intracellular adhesion molecule-1. Perhaps better design, chemical modifications and better drug delivery will improve the drug outcome.


In conclusion, there were high hopes that were raised for siRNAs in AMD about three years ago, but the failures of two of the siRNA studies are disappointing and could represent a setback to the future of siRNA in treatments of ocular diseases. Now, two failed major trials and hundreds of millions of dollars later, siRNAs are still stuck in the starting gate with not much to show for all the effort and money that were put into them. The future of siRNA in AMD probably lies today on the results of the MONET study. RP


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