857. A Versatile and Efficient Viral Vector Platform for Stable In Vivo Expression of Short Hairpin RNA

Top of pageAbstract A variety of non-viral and viral vectors are currently being developed for the in vivo expression of short hairpin RNA, with the goal to use these vectors for the suppression of cellular or viral transcripts involved in human pathogenesis. Amongst these potential shRNA delivery vehicles, double-stranded (ds) vectors based on Adeno-associated virus (AAV) hold particular promise, since they give very robust and long-term gene expression in the transduced cell, while lacking any inherent toxicity. However, a variety of problems associated with the current generation of dsAAV vector constructs hamper their broad evaluation. Firstly, the constructs are relatively unstable in bacteria, due to the fact they contain two almost identical viral DNA packaging elements (ITRs) in close proximity. Secondly, encapsidation of the vector genomes also yields low particle titers, which might again be related to the use of two homologous ITRs, likely increasing the risk for detrimental deletions in one of them. Thirdly, there is no dsAAV plasmid construct available thus far that would allow the simple and straight-forward cloning of shRNA expression cassettes. To overcome these hurdles, we have generated a set of novel dsAAV constructs with the following main characteristics : 1) the two ITRs are derived from two different AAV serotypes, which significantly increased plasmid stability, as well as viral particle titers, 2) three different basic plasmid versions were made, carrying a U6, H1, or 7SK promoter to drive shRNA expression, 3) each construct carries a unique restriction enzyme recognition site allowing the rapid and efficient cloning of any shRNA behind these promoters, 4) each plasmid moreover expresses a gfp reporter gene for easy detection of transduced cells, and 5) an additional large number of unique restriction sites allows for the convenient replacement of any vector component with custom sequences. Thus far, we have successfully used these constructs for the generation and packaging of over 20 different shRNA expression cassettes into dsAAV. We did not encounter any of the difficulties related to the original dsAAV vector design, in contrast, all of our clones were found to be stable in bacteria and did not accumulate mutations in the ITRs that would affect DNA replication or packaging. Encapsidation of the various constructs into capsids derived from multiple AAV serotypes was feasible and efficient, with final particle titers typically starting at 11012, and thus being identical to those obtained with genuine single-stranded AAV constructs. To this point, we have mainly used capsids from serotype 8 for dsAAV particle production, in line with our main interest, gene transfer to the liver. Analysis of gfp expression from the dsAAV vectors showed that low doses of 21011 dsAAV-8 particles mediated transduction of entire mouse livers. This allowed us to demonstrate complete and stable in vivo knockdown of transgenes expressed in the liver, most notably, of genes from Hepatitis B virus. We believe that the combination of our easy-to-manufacture and stable vector construct, together with the extreme efficiency of the final product, will help to establish dsAAV as a most powerful tool for in vivo shRNA expression.