Small-interference RNA technology: SIRNA 027, bevasiranib, and REDD14NP

John T. Thompson, MD and Quan Dong Nguyen, MD, MSc

CHAPTER

40

KEY FEATURES

The treatment of ocular diseases by controlling gene expression is a potentially powerful means of treating any retinal disease where a protein produced by cells is detrimental to the health of the organism. Posttranscriptional gene silencing occurs when the messenger RNA (mRNA) of a particular target gene is destroyed. The destruction of mRNA coding for the injurious protein prevents translation to form an active gene product. This chapter will discuss two new drugs, bevasiranib, which blocks and destroys mRNA coding vascular endothelial growth factor (VEGF) and SIRNA-027, which destroys mRNA coding for VEGF receptor. These two drugs are relatively new, such that efficacy and comparison with other agents, contraindications, ocular/ systemic complications, and drug interactions have not been studied; therefore, these data cannot be presented in any detail in this chapter. Relevant safety data will be presented in the description of the phase I and II trials for these two drugs. In addition, the chapter will also briefly discuss a third mRNA inhibitor, REDD 14NP, which is a small-inter- ference RNA (siRNA) that acts via RNA interference to inhibit the expression of the hypoxia-inducible gene, RTP801.

INTRODUCTION AND HISTORY OF SIRNA FOR RETINAL DISEASES

RNA interference (RNAi) is a common mechanism of posttranscriptional gene silencing. It was initially discovered in the segmented worm Caenorhabditis elegans by Fire and colleagues.1 They found that a doublestranded RNA homologous to a specific gene could turn off that gene. The process is initiated when a larger double-stranded RNA is cleaved by a ribonuclease named Dicer into short double-stranded fragments called siRNAs.

siRNAs are typically 21–23 nucleotides in length and will block protein production for specific proteins encoded by mRNAs whose target sequences are homologous to the siRNAs. Proteins are normally synthesized by a process where the DNA coding for a particular protein is transcribed into mRNA. The mRNA is then translated into protein. During the process of RNAi, siRNAs are incorporated into a multi­ protein endoribonuclease complex termed the RNA-induced silencing complex (RISC). Ahelicase within the RISC unwinds the duplex siRNA, allowing its antisense strand to bind mRNA with a high degree of sequence complementarity. An RNase within RISC then degrades the target mRNA by cleavage, which results in silenced gene expression and reduced protein production, as the cleaved target mRNA cannot reassemble. The same activated RISC can then bind to another identical target mRNA and the process may be repeated hundreds or thousands of times to amplify the response (Figure 40.1).

The prevention of protein synthesis has important implications in many diseases where abnormal production of proteins leads to a particular disease. Antibody and aptamer-based drugs are often designed to bind proteins that cause disease and inactivate them. In contrast to this mechanism of action, RNA interference prevents production of the protein by eliminating the mRNA.

There are many potential retinal diseases which can be targeted by siRNA technology. An obvious choice was to investigate the role of inhibition of VEGF for the treatment of choroidal neovascularization (CNV) since other drugs which block VEGF, such as ranibizumab, bevacizumab, and pegaptanib, were being developed.

Age-related macular degeneration (AMD) is a major cause of decreased vision in elderly patients and has two forms: neovascular (exudative or wet) and nonexudative (dry). Approximately 10% of patients develop the exudative form of AMD, characterized by CNV. The prevalence of either form of AMD is 7.9% in persons 75 years or older, and the presence of milder forms at baseline increases the risk of progression to more advanced forms of maculopathy.2,3

VEGF is an important stimulus for the development of CNV and inhibition of VEGF has been demonstrated to inhibit CNV in patients with AMD and improve visual outcomes when compared to the natural history of the disease.4,5 Ranibizumab, bevacizumab, and pegaptanib all require frequent intravitreal injections (every 4–6 weeks) for 1–2 years to obtain the best visual results.4,6–8 The burden of frequent injections and the desire to achieve more improvements in visual acuity has led to a search for other inhibitors of VEGF which may help in the treatment of CNV.

The application of siRNA technology for the treatment of CNV represents a new strategy to prevent VEGF synthesis. Several approaches for the inhibition of VEGF are under investigation. OPKO Health (formerly Acuity Pharmaceuticals: Miami, FL) has been developing bevasiranib (previously called Cand5), an siRNA targeting VEGF, to treat AMD. SIRNA Therapeutics (San Francisco, CA) has been evaluating SIRNA-027, an siRNA directed against VEGF receptor 1, in partnership with Merck and Allergan. Another siRNA targeting the VEGF pathway from Alnylam Pharmaceuticals was evaluated in preclinical studies but has not progressed to human trials. Quark has partnered with Pfizer to investigate the role of REDD14NP, the siRNA that inhibits the hypoxia-inducible gene, RTP801, which potentially prevents production of VEGF.

PHARMACOLOGY, DRUG MECHANISM, AND DRUG EFFECTS IN NONOCULAR DISEASES

The first report of the use of siRNA directed against VEGF was published by Reich9 in a model of CNV in mice, but its application to other diseases, specifically oncology, where VEGF plays an important role in the growth of tumors, was rapidly recognized.

siRNA directed against VEGF has shown positive effects in multiple cell culture and animal models of cancer. The cancer cells in which anti-VEGF therapy has shown positive effects include human fibro­ sarcoma cells, human prostate cancer cells, human gastric cancer cells, cutaneous malignant melanoma cells, human colorectal cancer cells, murine mammary cancer cells, murine squamous cell carcinomas, murine subcutaneous adenocarcinoma tumors, human leukemia cells, human breast cancer cells, human nasopharyngeal cancer cells, human osteosarcoma cells, and murine liver fibrotic cells.10–29 The exact