20. Directed Evolution of AAV2 Yields Novel Capsid Variants

Abstract

Adeno-associated virus (AAV) is a highly promising gene delivery vehicle, though gene transfer barriers can in cases limit its efficiency. These barriers arise at every step of delivery: the transit of the vector from injection to a cell surface, receptor binding and uptake, intracellular trafficking, and nuclear steps. The AAV gene transfer properties at each of these steps are determined by its capsid structure. Previous capsid modifications that alter AAV tropism, as well as the existence of multiple AAV serotypes, suggest that the structure-function relationships of the AAV capsid are reasonably plastic. We have taken advantage of this remarkable capsid plasticity to generate a large random mutant AAV library (1e6) and select for mutant AAV virions that can overcome several barriers to infection. Specifically, we have selected the AAV2 library for infectious particles with altered heparan sulfate (HS) affinity and for the ability to evade an AAV2 immune response. First, we have generated mutants with both lower and higher affinity to heparin, which could prove valuable in controlling the therapeutic zone of an AAV vector in tissues where ECM HS hinders AAV2 diffusion. Furthermore, we have generated vector variants that have greater than 100-fold improved resistance to human serum that neutralizes wild type AAV2, yet retain AAV2 gene delivery efficiency. These vectors may enable high gene delivery efficiency even in patients with pre-existing immunity, and the locations of point mutations on the capsid surface suggest new regions of functional importance to the virus. These AAV libraries therefore both provide useful variants for gene therapy applications as well as offer new avenues to dissect basic AAV biology.