adeno-associated virus Raw sequence reads

Short-hairpin RNAs (shRNAs) delivered by recombinant adeno-associated viruses (rAAVs) are valuable tools to study gene function in vivo and a promising gene therapy platform. Here, we show that incorporation of shRNA cassettes into rAAV constructs leads to vector genome truncation events independent of AAV serotype or short-hairpin DNA (shDNA) sequence. Furthermore, sequences with hairpin-like secondary structures including some elements commonly utilized in rAAV vectors (e.g. promoters) cause genomic truncations and heterogeneity. We demonstrate that these sequences drive the generation of intra-molecular double-stranded AAV genomes through a template-switching mechanism during vector replication. Finally, we have taken advantage of the newfound properties of shDNA sequences to functionally replace the inverted terminal repeat (ITR) sequence of conventional rAAV vectors, and have now created a new class of self-complimentary vectors. Together, our findings open doors to engineering a new generation of AAV vectors with improved properties.