Recombinant AAV as a platform for translating the therapeutic potential of RNA interference

Abstract

RNA interference has become a ubiquitous biological tool, and is being harnessed for therapeutic purposes as well. Therapeutic posttranscriptional gene silencing takes advantage of the endogenous RNAi pathway through delivery of either chemically synthesized siRNAs, or transgenes expressing hairpin-based inhibitory RNAs (e.g., shRNAs and artificial miRNAs). RNAi has expanded the field of viral gene therapy from gene replacement to gene knockdown. Here, we review various noncoding RNAs such as shRNAs, miRNAs, and miRNA decoys which can be utilized for therapeutic applications when expressed from recombinant adeno-associated vectors (AAV), and present examples of their basic design. In addition the basis of exploiting cellular miRNA profiles for detargeting AAV expression from specific cells is described. Finally, an overview of AAV-mediated RNAi preclinical studies is presented, and current RNAi-based clinical trials are reviewed.

Figure 1

RNAi and miRNA Biogenesis. Pri-miRNA are transcribed from either endogenous or vector-derived genes by RNA polymerase II or polymerase III. The pri-miRNA transcript has a 7-methylguanosine cap and poly-A tail, as it enters the microprocessor complex (Drosha & DGCR8) for its first cleavage event. The resulting pre-miRNA as well as vector-derived shRNAs can then be bound by the Exportin 5 RanGTP shuttle and exported though a nuclear pore into the cytoplasm. After export to the cytoplasm, pre-miRNAs and shRNAs associate with Dicer and the double-stranded RNA-binding protein TRBP. This cleavage event results in the production of ~22-nt-long miRNA/miRNA* duplex. The guide strand of the duplex is then preferentially loaded into Ago, whereas the passenger strand is usually degraded. The catalytic activity of the RISC is imparted by the Ago proteins 1–4, shown here is Ago2 which contains a PIWI domain with “slicer” activity.

Figure 2

RNAi tools that can be expressed from an adeno-associated vectors (AAV): main features and structure. (a) Structure of shRNAs and artificial miRNAs. The mature shRNA or miRNA guide strand is represented in red with the seed sequence in bold. The loop is represented in blue, in the miRNA the arrows mark the Drosha cleavage sites, adapted from Borel et al., 2011. (b) Representation of the endogenous mouse miR-122, where mature miRNA-122-5p is represented in red with the seed sequence in bold, and the mature miRNA-122-3p is represented in green; the arrows mark the Drosha cleavage sites. (Note in this case the guide strand is in 5p and the passenger strand is in 3p, depending on the thermostability of the strands this can be reversed in other miRNAs.) (c) Representative sequence and structure of a miRNA tough decoy (TuD). In red is the sequence that is complementary to the target miRNA (e.g., miR-122) adapted from Xie et al., 2012. (d) Illustration of a vector construct with four copies of target sequence for miR-142 at the 3′UTR of the cDNA, adapted from Brown et al., 2006.