Table of Contents 21-1

Therapeutic RNAi for Genetic Skeletal Disease?

RNA interference (RNAi) is a gene silencing phenomenon first identified in the nematode, C. elegans, but was subsequently found to occur in higher organisms including humans. It probably evolved as an ancient defense mechanism for cells to fend off mobile genetic elements, such as RNA viruses and transposons, but today it has been implicated in a growing number of cellular processes.

As discussed by Stevenson, RNAi involves sequence specific degradation of target RNAs triggered by the formation of double stranded RNA (dsRNA). When it occurs naturally, long dsRNA is processed to short interfering RNAs (siRNAs) 21-24 bases in length by a dsRNA-specific endonuclease named Dicer (Figure). They are incorporated into a nuclease complex referred to as the RNA-induced silencing complex or RISC. Unwinding of the siRNAs activates and directs RISC to the target RNAs, which are cleaved and degraded. The complementarity between the siRNA and the target RNA determines the sequence specificity of RNAi.

An important advance in the RNAi field was the discovery that exogenous synthetic siRNAs or endogenously synthesized siRNAs driven by viral vectors could be incorporated into RISC and induce sequence-specific degradation of target RNAs. This created an extremely powerful tool for scientists to “knock down” expression of genes of interest simply by adding synthetic RNA duplexes to the medium of cultured cells, introducing viral vectors that express siRNAs into cells or even generating transgenic animals that synthesize siRNAs.

RNAi is much more complex than outlined here, and there are many technical difficulties that complicate the use of RNAi to knock-down gene expression in experimental systems. Nevertheless, RNAi has stimulated considerable interest in the pharmaceutical/biotech industry as a potential therapeutic agent for human disease. The best examples to date have to do with treatment of infectious diseases, such as those caused by HIV, hepatitis viruses and poliovirus, as well as cancers that are mediated in part by overactive oncogenes. In the case of viral infections, interfering RNAs could be targeted to viral transcripts required for viral replication or survival. In the second case, using RNAi to silence expression of BCR-ABL, the fusion gene that results from the Philadelphia chromosome translocation in chronic myelogenous leukemia or mutated RAS oncogenes that drive several types of cancer would be appealing.

Receiving less attention to date, but of probably at least as much interest to readers of GGH, is the potential use of RNAi to knock down expression of mutant alleles in dominantly inherited genetic disease. In concept, siRNAs could be tailored to distinguish mutant from normal (wild type) alleles and block only mutant allele expression. This could convert a dominant negative disorder, ie, a disorder in which the product of the mutant allele interferes with the function of the normal (wild type) allele product, to a disorder that results from haploinsufficiency or functional loss of one allele. For families in which both forms occur, manifestations are usually milder in the form resulting from haploinsufficiency, ie, osteogenesis imperfecta type I – haploinsufficiency vs osteogenesis type II – dominant negative. Thus, there is potential benefit from this therapeutic strategy.

Despite the excitement and promise of therapeutic RNAi, there are many obstacles, the greatest of which is delivery. Systemically delivered siRNAs face degradation by nucleases, and the use of viral vectors to target organs of interest is still in its infancy. A recent publication by Soutscheck and colleagues provides evidence that chemically modified siRNAs can successfully knock down endogenous genes in living mice. More specifically, they targeted expression of the gene encoding apoprotein B (apoB) in the mouse liver and jejunum where it is known to be expressed at high levels with 2 siRNAs known to silence apoB in cultured cells. They modified the apoB siRNAs by chemically stabilizing their backbone and also by adding cholesterol to their 3’ end. The modified siRNAs were then compared to unmodified apoB siRNAs and other controls.

The results showed that the cholesterol-conjugated apoB siRNAs were significantly more stable in serum than their unconjugated counterparts. When administered intravenously, one of the conjugated apoB siRNAs was very effective at lowering apoB mRNA and apoB protein levels, as well as total cholesterol and LDL cholesterol. They observed no evidence of “off-target” effects, that is, effects attributed to silencing of genes other than apoB or other obvious complications from the injections. The authors concluded that exogenously administered chemically modified siRNAs can potentially be used to silence expression of endogenous genes involved in human disease.

Stevenson M: Therapeutic potential of RNA interference. N Eng J Med 2004;351:1772-7.

Soutschek J,Akinc A, Bramlage B, et al. Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature 2004;432:173-8.

Editor’s Comment: RNAi has had a major impact on science since its relatively recent discovery. It is still not entirely clear how it works and there remain concerns about specificity and the so-called off target effects on genes other than specifically targeted genes. Nevertheless, it has great promise as a means to treat not only cancer and infectious diseases, but genetic diseases in which mutant alleles differing from their normal alleles by only a single base can be specifically targeted. It will probably be years before such treatment becomes realistic for humans, but the success of substantially knocking down apoB expression by systemically administering chemically modified apoB siRNAs in mice is very encouraging. One note of caution is that the growing skeleton may be difficult to target because the cartilaginous growth plate is relatively avascular compared to most tissues such as liver and gut.

William A. Horton, MD