742. Molecular Evolution of Adeno-Associated Viral (AAV) Vectors Via DNA Family Shuffling of Primate and Non-Primate Serotypes (2006)

Gene transfer vectors based on naturally occurring serotypes of Adeno-associated virus (AAV) are gaining increasing attention due to their numerous advantages over other systems, including safety and capability to transduce a wide variety of cell types and tissues. However, remaining problems with AAV vectors include the high prevalence of neutralizing antibodies to at least some of the wildtype viruses in humans, and the fact that many relevant cell types are largely refractory to most AAV serotypes. Several strategies were proposed to overcome these hurdles, such as peptide insertion into the AAV-2 capsid or its site-directed mutagenesis, but required knowledge of the capsid structure and were thus restricted. Here, we pursued a most powerful approach to functional diversification of the AAV virion, by in vitro recombining the capsid genes of eight divergent primate and non-primate serotypes. In detail, we “shuffled” the capsid genes of AAV serotypes 2, 4, 5, 8 and 9, as well as of avian, bovine and caprine AAV, by DNase-fragmenting the respective sequences, followed by primer-less PCR amplification. The resulting hybrid capsid genes were fused with the rep gene and cloned between the ITRs from AAV-2, thus yielding a library of 3x104 shuffled AAV variants. Sequencing of 24 individual clones confirmed that each capsid gene consisted of domains from at least 2, and often 6 or more, distinct serotypes. Library functionality was initially demonstrated by its successful sequential amplification in kidney, liver and fibroblast cell lines. Our main goal was however to isolate novel AAVs from the library, able to efficiently transduce hepatocytes or entire livers in the presence of neutralizing anti-AAV antibodies. We therefore subjected the virions to a respective selection pressure, by mixing them with high doses of human sera containing such antibodies, prior to inoculation on human Huh-7 liver cells together with helper Adenovirus. Following five consecutive amplification rounds under these conditions, we indeed succeeded in isolating a shuffled virion able to escape serum inactivation, while maintaining a high potential for liver cell infection. Sequence analyses showed that this mutant consisted of domains from serotypes 2, 8 and 9, with the latter two particularly dominant on the exterior of the capsid, and AAV-2 sequences prevailing in the VP1 protein N- terminus. A direct comparison of vectors based on this hybrid with genuine serotypes 2, 8 or 9 showed that the novel clone was indeed at least 5-fold more efficient on Huh-7 cells, and also the only one next to AAV-8 able to escape serum inactivation, thus exemplifying the power of our approach. We therefore extended our strategy to even more challenging targets, primary human hepatocytes and intact murine livers. At this point, we have isolated a subset of shuffled AAV virions consisting of sequences from serotypes 2, 4, 8 and 9, and mediating efficient infection of these targets. Ongoing long-term in vivo studies will resolve the potential of the novel capsids for liver transduction, and will hopefully identify them as promising candidates to replace or complement genuine AAV serotypes in clinical vector applications.