Towards in vivo application of RNA interference - new toys, old problems

Abstract

RNA interference (RNAi) is the sequence-specific degradation of mRNA by short double-stranded RNA molecules. The technology, introduced only 5 years ago, has stimulated many fantasies regarding the future of functional gene analysis and gene therapy. Given its ease of application, its high efficiency and remarkable specificity, RNAi holds great promise for broad in vitro and in vivo application in all areas of biomedicine. Despite its potential, the major obstacle to the use of RNAi (as for all previous gene silencing approaches) is the need for efficient and sustained delivery of small interfering RNA into primary mammalian cells, and specific targeting of particular cell types in vivo.

Figure 1

The RNA interference pathway. Long double-stranded RNA (dsRNA) or small hairpin RNA (shRNA) is processed by Dicer to form a small interfering RNA (siRNA), which associates with RNA-induced silencing protein complex (RISC) and mediates target sequence specificity for subsequent mRNA cleavage. (See text for further details.)

Figure 2

Approaches to endogenous expression of siRNAs in mammalian cells. (a) Sense and antisense strand of the siRNA duplex are expressed from separate promoters. (b) siRNA duplex is expressed as a stem-loop structure (small hairpin RNA [shRNA]) from a single promotor. Sense and antisense strands are separated by a loop-forming spacer. The construct is further processed by Dicer within the cell to form a functional siRNA. In both cases transcription is terminated by six consecutive thymidine residues.