744. Novel Vectors for Gene Therapy: Covering the Spectrum of Primate AAV Diversity

Abstract

Top of pageAbstract One strategy to enhance the performance of AAV vectors is to evaluate the impact of capsid variation present in natural isolates of AAV virus. Indeed, one of the first AAVs recovered by our recent screen of novel AAVs, called AAV serotype 8, demonstrated substantially improved gene transfer in liver. Further building on this theme, we generated a catalog of AAV vectors that capture the entire spectrum of natural capsid variation and have screened these vectors for gene transfer to a variety of targets. A total of 30 vectors were generated and optimized by site-directed mutagenesis and tested in vivo in liver and muscle. Minor variations in capsid sequence were found to impact gene transfer applications dramatically. In order to pinpoint domains or residues that correlate with suboptimal vector production, or in vitro or in vivo gene transfer, we performed an extensive structure-function analysis. This highlighted a class of residues on existing capsid sequences that were found to be of particular interest. Atypical residues in otherwise entirely conserved regions consistently correlated with a loss in vector performance. These residues, termed singletons, were reverted back to their conserved state. Both the original and the variant were compared in their ability to package and perform in vitro and in vivo in C57BL/6 mice. Expression of the reporter gene hAAT was used to measure gene transfer efficiency. Dramatic improvement as a result of singleton modifications was observed. Generally in vitro transduction was mildly improved with few exceptions. Singleton modification resulted in improved in vivo gene transfer to liver and muscle. There were few examples however where in vivo performance was decreased, highlighting the critical importance for tropism of these vectors. Interestingly, AAV6 was evaluated before and after removal of two singleton residues. Residue K531 was found to be responsible for the superior tropism in both liver and muscle when compared to the closely related AAV1. Data on 30 AAV based gene therapy vectors of a variety of serological profiles, isolated from 5 primate species and representing all AAV clades will be presented. Evaluation for gene transfer in muscle identified 10 vectors superior to or as efficient as AAV8. Several lead candidates have been identified for liver directed gene therapy, a subset of which is to be tested in non-human primates. This study approaches the development of second generation vectors using an approach more like that used to identify small molecule pharmaceutics. We start from a natural library of AAV gene transfer vehicles. Based on correlates of functionality with structure, the particle is then engineered into lead molecules for the tissue target of interest. It is deemed critical to progress several candidates through pre-clinical development, in order to overcome hurdles like species difference of gene transfer, serological burden of a target population and other forms of pre-existing immunity.