Lentiviral-mediated delivery of siRNAs for antiviral therapy

Abstract

Lentiviral vectors portend a promising system to deliver antiviral genes for treating viral infections such as HIV-1 as they are capable of stably transducing both dividing and nondividing cells. Recently, small interfering RNAs (siRNAs) have been shown to be quite efficacious in silencing target genes. RNA interference is a natural mechanism, conserved in nature from Yeast to Humans, by which siRNAs operate to specifically and potently down regulate the expression of a target gene either transcriptionally (targeted to DNA) or post-transcriptionally (targeted to mRNA). The specificity and relative simplicity of siRNA design insinuate that siRNAs will prove to be favorable therapeutic agents. Since siRNAs are a small nucleic acid reagents, they are unlikely to elicit an immune response and genes encoding these siRNAs can be easily manipulated and delivered by lentiviral vectors to target cells. As such, lentiviral vectors expressing siRNAs represent a potential therapeutic approach for the treatment of viral infections such as HIV-1. This review will focus on the development, lentiviral based delivery, and the potential therapeutic use of siRNAs in treating viral infections.

Figure 1

RNAi pathways in mammalian cells. RNAi can operate at the transcriptional and post-transcriptional level or possibly a combination of both and is based on the specific targeting of siRNA or shRNAs to an mRNA or a promoter. A cell can be stably transduced with a lentiviral vector that expresses siRNAs either from two independent promoters (U6 Pol-III) or a single U6 Pol-III promoter driving the expression of a hairpin shRNA targeting a particular gene of interest. (1–2) The expressed siRNA or shRNAs may be processed by Drosha and subsequently exported by Exportin 5.^, Once exported out of the nucleus they (2) are handed off to Dicer which then processes them into 21 base siRNAs (3) and the antisense strand loaded into RISC, ultimately leading to slicing of the target mRNA (4). Alternatively, siRNAs can function in a TGS based manner following expression from the lentiviral vector (A) the siRNAs get bound by a complex that also contains DNMTs (K. Morris personal observation) (B), which can interact with histone deacetylase (HDAC) and the histone methyltransferase (SUV39H1) to essentially replace the acetate group of histone 3 Lysine 9 with a methyl group subsequently silencing the targeted promoter in a chromatin modifying based pathway (C) which may or may not result in DNA methylation of the targeted promoter. Finally, it is possible that the promoter directed siRNAs might be expressed and exported out of the nucleus, loaded into RISC in the cytoplasm and get shuttled back to the nucleus, possibly by an argonaute related pathway, where they can function to suppress gene expression through an argonaute protein mediated mechanism.

Figure 2

Lentiviral vector delivery and expression of shRNAs targeting HIV-1. Lentiviral vectors can stably transduce target cells (1) integrate and constitutively express anti-HIV-1 siRNAs or shRNAs (shRNAs are shown) (2). The anti-HIV-1 shRNAs are exported via the Exportin 5 pathway subsequently delivering the shRNA to Dicer (3) where the loop is removed by Dicer processing and the correctly processed shRNA, specifically the antisense strand loaded into RISC, where the HIV-1 mRNA is targeted and degraded (4). The lentiviral vector transduced cells can be used to either protect a cell from viral infection (A–C, depicts viral entry-integration while (D) depicts viral mRNA production from the provirus) or therapeutically to reduce overall viral burden on a cell that has already been infected.