Interfering with RNA: Ready for Prime Time - RNAi

Dr. Fitzgerald also says addition of RNAi technology has helped uncover ?several important mechanisms and target proteins for compounds that, while they had unique therapeutic activities in animal models, turned out to have different molecular targets than we originally thought. Early hints from expression profiling showed that even if a small molecule and an siRNA hit the same targets, they have quite different effects.?
For example, he points out, a small molecule may inhibit the activity of one particular protein, such as a tyrosine kinase, affecting its target within an hour. But an siRNA will remove all of the target protein from a cell by eliminating the mRNA from which it is translated, an effect that can take up to 48 hours to occur.
With regard to unanticipated effects, an siRNA to a typical transmembrane tyrosine kinase will not only remove the tyrosine kinase activity of that specific kinase but may result in the disruption of several protein complexes bound to the receptor, or other kinases in that family. A small molecule may result in the loss of only one kinase activity.
?These are different processes with different kinetics,? Dr. Fitzgerald cautions, ?So experiments comparing the phenotypes resulting from them are required for drug effect validation.?
Interestingly, Dr. Fitzgerald points out that cell-based assays might come back into fashion. Most companies operate in an ultra-high throughput mode, screening millions of compounds against an isolated, purified target with the hope that when inhibited in the more complex environment of a cell, a potential drug will have therapeutic activity.
But, he says, these methods are unlikely to uncover drugs that are effective because they impact multiple targets, counteracting the fail-safe mechanism inherent in biological systems. He believes that the combination of better cell biology and siRNA may help in this regard.
The reason industry moved away from cell-based screening, was the difficulties inherent in uncovering molecular targets of the drugs discovered in this fashion. These new technologies now make this easier.
siRNAs as Therapeutics
While companies that presented at ?RNAi for Pathway Analysis? focus on RNAi technology for drug discovery, development of siRNAs as potential therapeutics has moved ahead.
Drug developers cite the need to overcome several obstacles to using these molecules as drugs, including delivery, appropriate targeting, stability, and safety. Already in Phase I trials with siRNAs targeted at age-related macular degeneration (AMD) are Acuity Pharmaceuticals with its Cand5 siRNA drug and Sirna Therapeutics with its siRNA-07. Both drugs target VEGF targets, and are designed to shut down the pathological angiogenesis associated with AMD.
Alnylam Pharmaceuticals also plans to enter the clinic with their siRNA candidate molecule for AMD in the second half of 2005. Reasons for the popularity of this target include a well-validated drug target (VEGF), relative ease of access to the eye, and previous clinical experience and success with ocular antisense drugs, as well as the fact that a successful AMD drug would meet a significant unmet medical need.
According to Zachary Zimmerman, Ph.D., Alnylam?s director of external alliances, the company has addressed several issues impacting the use of siRNAs as drugs. ?We know that certain sequence motifs in the siRNAs stimulate the immune system, so we avoid these motifs when we make our drug candidates,? he says. ?We test a fairly large number of molecules in vitro, and always come up with a handful that will work well without unwanted stimulatory effects.?
Further, Alnylam scientists developed molecular modifications that get around siRNA delivery and stability issues. In the November 2004 issue of Nature, company scientists described siRNAs stabilized with partial phosphorothioate backbones and with 2'-O-methyl sugar modifications on the 3' end to prevent their degradation by endogenous enzymes.
Conjugation with cholesterol facilitated siRNA uptake in target cells of transgenic mice expressing human apoB lipoprotein, resulting in decreased plasma levels of apoB and reduced total cholesterol at a level similar to cholesterol-lowering statins.
The company also recently described results of preclinical in vivo studies showing that a single low dose of the company?s Aln-RSV01 siRNA drug effectively treated respiratory syncytial virus (RSV) infection in mice and protected them against subsequent infection.
Directed against an RSV gene required for viral replication, the siRNA was administered intranasally. Alnylam is developing a formulation of the compound to allow its direct introduction to the lung by aerosolization through a nebulizer.
Alnylam has two alliances with Merck, including one for the development and commercialization of its AMD drug, licensing agreements with Isis Pharmaceuticals, and a partnership with Medtronic, as well as collaborations with Mayo Clinic and the Cystic Fibrosis Foundation.
According to Dr. Zimmerman, ?We are interested in direct RNAi therapeutics to treat CNS diseases, and we realize that a drug-device combination will be required for delivery. Partnering with a leading medical device company such as Medtronic will help overcome the CNS delivery challenge.?
Patricia F. Dimond, Ph.D., is president of Dimond Biotechnology Consulting, consultants to the biotech industry in investor development and strategic planning and communications. Phone: (508) 255-0221. E-mail: drpdimond@comcast.net