Directed evolution of AAV capsids for improved efficacy and specificity of delivery topreclinical models of human liver

Abstract

Introduction and objectiveRecombinant vectors derived from adeno-associated viruses (rAAVs) are the leading platform in human gene therapyapplications, with high-profile examples targeting diseases of the central nervous system, eye and liver. The liver, quitelikely a natural host organ for wild type AAV2, is a particularly attractive target for the development of AAV-mediatedgene therapies. Despite large number of AAV variants current at various stages of development as carriers of livertherapeutics, thus far no liver-directed AAV-based therapy has obtained market authorization. Strong preclinical datais the cornerstone of any translational program, and while AAV bioengineering is commonly applied to try to developnovel human-tropic vectors for clinical applications, due to species-to-species differences, dedicated vectors to su-pport preclinical work may need to be developed. Here we applied AAV directed evolution and in vitro selection toidentify AAV capsids that target human liver cells in vitro.Material and methodsUsing DNA shuffling technology, we have generated a capsid gene library based on natural parental serotypes (AAV1through AAV12). Shuffled capsid library was selected in a preclinical model of human liver.ResultsThe AAV variants enriched based on their improved efficiency of transduction of a human hepatocyte cell line werevectorized and subsequently functionally characterized on human cell lines. This directed evolution method enabledus to select novel AAV variant, AAV-CH4.2. While the selected variant did not exceed the parental serotype in termsof transduction efficiency, it was substantially more efficient at packaging than its closest homolog, serotypes AAV6.ConclusionsBased on its strong transduction profile and manufacturability, we believe that AAV-CH4.2 is a strong candidate forfurther evaluation and as a potential novel gene therapy vector for preclinical studies in human liver applications.