In contrast to miRNAs, siRNAs are usually perfectly complementary to their targets. They are thus very effective at eliminating gene expression. For this reason, synthetic siRNAs have been generated for a number of therapeutic uses. But of course, there are hurdles: stability, potency, off-target effects, and efficient delivery of synthetic siRNAs are some of the major challenges for successful application of this technology in the clinic.
Quark Pharmaceuticals is committed exclusively to developing siRNA drugs. One of its siRNAs has finished Phase II trials for the treatment of wet age-related macular degeneration and diabetic macular edema.
This siRNA was licensed to Pfizer, which conducted the trials. The diabetic macular edema trial was reportedly the first siRNA Phase II trial to show dose-dependent efficacy—there was an actual and significant improvement in clinical endpoint (visual acuity), not just a knockdown of gene expression.
Elena Feinstein, M.D., Ph.D., Quark’s CSO, spoke about some exciting results with another of the firm’s therapeutic siRNAs, this one to treat nonarteritic ischemic optic neuropathy, at Keystone Symposium’s recent “Nucleic Acid Therapeutics” conference. She characterized these results as “fresh from the oven.”
Both siRNAs treat eye diseases; Dr. Feinstein said that they decided to start there because there was no delivery problem. Quark uses a chemical modification of its siRNAs to render them highly specific and stable to nucleases, and to prevent their generating an immune response.
“These modifications are suitable for eye use. You can’t inject liposomes or viruses into the eye—it will cause inflammation,” said Dr. Feinstein. “But ours can be used for other local administration.”
The new results she presented show that when given their new siRNA, “these patients did not deteriorate further. From the moment they received the injection, visual loss was halted in all 20 patients. In fact, all of them gained vision that lasted over the three months of analysis.”
Dr. Feinstein noted that this was a Phase I trial to assess safety, and that there were no placebos, since that would involve an injection into the eye that no one was particularly keen to undergo needlessly. But even so, she was excited because historical data in the same type of patients shows further deterioration of visual acuity occurring over time in many of them.
Dicer is an enzyme that dices double-stranded RNA and pre-microRNA into siRNA fragments and facilitates the formation of the RISC. The aptly named Dicerna Pharmaceuticals takes advantage of Dicer by linking a Dicer substrate sequence to its siRNA molecules so that they are more efficiently loaded into the RISC.
Darren Wong, Ph.D., senior director of research at Dicerna, pointed out a number of benefits to this loading. “We made an asymmetric molecule that forces it into Dicer in a particular orientation, so they are more potent than first-generation siRNA molecules, which don’t normally engage Dicer,” Dr. Wong said. “They can also avoid immunogenic proteins.”
Dicerna has been focused on delivering its Dicer Substrate siRNA (DsiRNA) molecules to the liver. Dr. Wong and Bob Brown, Dicerna’s CSO, explained that these DsiRNAs can penetrate not only healthy livers but fibrotic and cirrhotic livers, where tumors are often found. They can thus be used to treat other liver diseases besides cancers.
These DsiRNAs have also been successfully delivered to prostate tumors when implanted into animals. Dicerna has the ability to extensively chemically modify and/or link DsiRNAs directly to other classes of pharmaceutical molecules, such as aptamers, peptides, antibodies, and small molecules. This ability can improve biodistribution and cellular delivery, thereby achieving a more desirable pharmacokinetic profile.
Plants use siRNAs to combat viral invaders. Ruth Broering, Ph.D., and her colleagues in the department of gastroenterology and hepatology at the University Hospital of Essen, Germany, has shown that perhaps one day we can, too. Dr. Broering used siRNA to knock down the expression of ISG15 (interferon stimulated gene 15), a proviral factor for hepatitis C replication and a regulator of the interferon response.
In both tissue culture and in mice, siRNA boosted the responsiveness to interferon. Its direct suppression of hepatitis C replication to a degree commensurate with that achieved by interferon therapy has been demonstrated for different hepatitis C cell culture systems, and they hope to prove this antiviral concept soon in vivo using mouse models susceptible to hepatitis C virus infection.
Their findings suggest that ISG15 knockdown may be used therapeutically for those patients who do not respond to interferon therapy. As it turns out, those patients tend to have high ISG15 expression.
The siRNA Dr. Broering used was provided by the now defunct company Roche Kulmbach. It is nanolipid-formulated and has ribose-modified nucleotides (2´O-methyl or 2´-fluor) that eliminate the innate immune response siRNAs can elicit; it manages this by avoiding Toll-like receptor activation.
Stephen Hart, Ph.D., a reader in molecular genetics at the Wolfson Centre for Gene Therapy of Childhood Diseases at University College, London, didn’t choose a target and go searching for the optimal delivery system. Rather, he develops delivery systems, and then applies them to the appropriate target organs.
He is developing siRNA therapeutics for cystic fibrosis to be delivered to the lung via nebulization, siRNA therapeutics for neuroblastoma to be delivered via systemic delivery, and siRNA therapeutics for neurodegenerative disease to be delivered via injection into the brain.
All of his formulations consist of a liposome and a targeting peptide that electrostatically self-assemble into complexes when mixed with an siRNA. Dr. Hart noted, “The formulation is stable. It forms stable nanoparticles of an appropriate size, and the siRNAs are protected from nucleases and can be released into the cytoplasm where they can access the RISC.”
The charge of the nanocomplex can be controlled by varying the nature and ratio of the contents. For delivery to the lung the nanoparticles are cationic, but Dr. Hart noted that for other applications different biophysical properties may be required—for example, “cationic particles are no good for systemic delivery, because they will bind to serum proteins.”
siRNA has immense potential as a therapeutic reagent, especially for those diseases that have a well-characterized but nondruggable target gene. Delivery to in vivo targets has been one of the major challenges hampering progress in their clinical development. Yet a few different solutions, such as those highlighted in this article, are bringing these molecules closer and closer to being utilized as drugs.