1085. Directed Evolution of AAV2 Leads to Improved Viral Function

Abstract

Adeno-associated viral (AAV) vectors are extremely safe and efficient gene delivery vehicles. However, numerous challenges in vector design remain, including neutralizing antibody responses, tissue transport, and infection of resistant cell types. In each case, the AAV capsid structure determines how the vector performs when presented with these barriers. Nature has evolved multiple AAV serotypes using mutagenesis and recombination, indicating a natural level of plasticity in viral structure and function. We have employed analogous techniques to rapidly generate a large random mutant AAV2 library and have selected for mutant AAV virions that display novel phenotypes. Specifically, we have selected the AAV2 library for infectious particles with altered heparin sulfate (HS) affinity and for the ability to evade an AAV2 immune response. Characterization of the mutants with both lower and higher affinity to heparin led to the location of novel point mutations on the capsid structure. Several of these mutations lie inside the capsid while two exposed mutations (K258E and S261F) lie in the canyon region. Furthermore, we have evolved vector variants that have greater than 100-fold improved resistance to serum that neutralizes wild type AAV2, yet retain AAV2 gene delivery efficiency. Analysis of the point mutations revealed one change on the surface of the 3-fold spike (N587I) and another change on the 2-fold dimple region (T716A) were critical to the enhanced resistance. Testing these vectors in animal studies may indicate that they could mediate gene delivery even in patients with pre-existing immunity. Finally, our libraries can be further analyzed to isolate mutants that surpass other barriers to successful gene delivery.