Small RNA molecules, processed from larger RNA molecules, play roles in the regulation of gene expression by binding to target genes. Small interfering RNAs (siRNAs), generally 21–25 nucleotides in length, bind to target mRNA, probably through perfect (or close) base-pairing, and initiate sequence-specific cleavage. MicroRNAs are small, endogenous RNAs, usually 19–25 nucleotides in length that are cleaved from hairpin structures. Hundreds of microRNAs have been identified, and each is thought to have hundreds of target genes. They regulate these target genes by inhibiting the translation and processing of transcripts, probably through imperfect base-pairing. MicroRNAs are involved in normal development processes, including cellular growth, cell differentiation, and programmed cell death. MicroRNAs also play a role in disease processes, including tumor development, age-related disease, and disorders of the immune system, cardiovascular system, and nervous system.
RNA interference (RNAi)—a mechanism of gene silencing via these small RNAs that was originally described in plants and invertebrates—is known to occur in mammalian cells, and therapeutic applications of RNAi using both siRNA and microRNA are being developed. Some applications are based on the knowledge that RNAi is used in some organisms to defend against viruses and genetic “invaders” (e.g., transposable elements), and that synthetic siRNAs can be active in RNAi in mammalian cells. Among the remaining challenges that need to be addressed are techniques for determining the correct sequence for the therapeutic RNA, delivering that RNA to the appropriate site, and protecting it from rapid degradation.
Topical ProtectionDeborah Palliser, Assistant Professor of Microbiology and Immunology at the Albert Einstein College of Medicine in the Bronx, NY, and Judy Lieberman, Professor of Pediatrics at Harvard Medical School in Boston, MA, recently published the results of their collaborative study in Cell Host & Microbe. They showed that a topical microbicide preparation silencing two genes via RNAi conferred long-lasting protection against genital herpes caused by herpes simplex virus 2 (HSV-2) in a mouse model. The two genes targeted by siRNA were UL29, a viral gene required for replication; and the gene for nectin-1, a protein on the surface of vaginal cells that is used by HSV-2 to enter those cells. The viral gene suppression is immediate, but short-lived, lasting only ∼1–2 days. Though the nectin-1 cell-surface receptor gene is not silenced until several days after application, viral challenge indicated that its suppression gave protection for 1 week. Topical application of a solution containing the therapeutic construct is protective if administered 1 week before to a few hours after exposure to HSV-2. The microbicide does not cause inflammation of vaginal tissue, an effect seen with other siRNAs.
Palliser started this work while studying for her Ph.D. in Lieberman's laboratory. Now, she is using questions that arose during her time as a post-doc to influence her research at Einstein. She says that toxicity of siRNAs has been an issue, as well as the fact that the molecules are incredibly short-lived. Lipid complexes have been used to increase longevity of siRNA, but some of these complexes, despite their longevity, still have toxicity issues. As shown in Figure 1, application of the transfection lipid reagent oligofectamine resulted in a small, but significant influx of CD45+ inflammatory cells. Palliser says, “This was one of the early findings that prompted us to investigate methods of siRNA delivery that did not require lipid reagents and lead us to using the cholesterol-conjugated siRNAs. The most exciting and significant questions for therapy are [about how] to have as few side effects as possible with long-lived protection.”
One follow-up project Palliser is pursuing is targeted delivery of siRNA to specific cells types to restrict side effects. One of her interests is dendritic cells, which play a role in the activation of immune responses to microorganisms invading mucosal surfaces. Another issue to investigate is what the long-term effects of downregulation of host cell genes (e.g., nectin-1) will be. Nectin-1 is involved in the formation of tight junctions, so the use of an anti-HSV-2 therapeutic that disrupts tight junctions on a long-term basis might not be good. However, tissues to which this therapy is applied are sloughed off on a cyclical basis, Palliser explains, so side effects may be limited. Possible therapeutic applications include use as a prophylactic to infection or to treat reactivation of the virus in already-infected individuals.
