Lentiviral delivery of short hairpin RNAs

Abstract

In less than a decade after discovery, RNA interference-mediated gene silencing is already being tested as potential therapy in clinical trials for a number of diseases. Lentiviral vectors provide a means to express short hairpin RNA (shRNA) to induce stable and long-term gene silencing in both dividing and non-dividing cells and thus, are being intensively investigated for this purpose. However, induction of long-term shRNA expression can also cause toxicities by inducing off-target effects and interference with the endogenous micro-RNA (miRNA) pathway that regulates cellular gene expression. Recently, several advances have been made in the shRNA vector design to mimic cellular miRNA processing and to express multiplex siRNAs in a tightly regulated and reversible manner to overcome toxicities. In this review we describe some of these advances, focusing on the progress made in the development of lentiviral shRNA delivery strategies to combat viral infections.

Fig. 1. Schematic of non-replicating lentiviral vector for stable shRNA expression

Suitable host cells (such as 293T cells) are trasfected with a mixture of plasmids consisting of i) an shRNA expression cassette, ii) a packaging cassette and iii) a heterologous (VSV-G) viral envelope expression cassette. The generated lentivirus is then used to transduce the desired cell type for shRNA expression. Because only the vector containing the shRNA expression cassette (devoid of the viral structural genes) integrates into the host cell genome in the transduced cells, shRNA is continually expressed but infectious virus is not produced.

Fig. 2. Biogenesis of conventional and miRNA-based shRNA

The left panel shows the biogenesis of the Pol III promoter-driven conventional shRNAs (originally developed without the knowledge of miRNA biogenesis). Here the transcript only contains the hairpin RNA sequence that is directly exported to the cytoplasm. The middle panel indicates the natural miRNA biogenesis. The primary miRNA transcript contains the mature miRNA hairpin as well as a long flanking sequence and is processed first by Drosha into pre-miRNA that is then exported to the cytoplasm for Dicer processing. The right panel shows miRNA-based shRNA expression. Here the shRNA construct (controlled by either Pol III or Pol II promoter) is designed to mimic the pri-miRNA by including the miRNA flanking sequence into to shRNA stem. The resulting transcript is processed like miRNA by Drosha, followed by Dicer into siRNA. Although during miRNA biogenesis, both strands are sometimes selected, selection of the passenger strand should be avoided in the shRNA design to reduce off-target effects.

Fig. 3. Rational design of shRNA based on the current knowledge about Drosha and Dicer processing

Drosha cleaves the pri-miRNA ~11bp from the ss-ds RNA junction irrespective of whether the mature miRNA (marked in red) is derived from the 5′ (A) or the 3′ (B) arm of the hairpin. The miRNA stem also generally contains internal mismatches ~12nt from the 5′ end of mature miRNA that might help avoid abortive processing. Thus for the design of shRNA depicted on the right, 20–40 nt flanking sequence and an internal bulge as depicted should be included to ensure accurate Drosha processing. Also the shRNA terminus should be designed to favor the selection of guide vs passenger strand by incorporating mismatches or AU at the 5′ end as depicted.