New Methods in Engineering Adeno-Associated Virus (AAV) for Improved Gene Delivery

Gene delivery vehicles, or vectors, based on adeno-associated viruses (AAV) have demonstrated success in both preclinical disease models and recently in human clinical trials for several disease targets, including muscular dystrophy, hemophilia, Parkinson's disease, Leber's congenital amaurosis, and macular degeneration. AAV has multiple characteristics that make it an effective gene therapy vector: the parent virus is nonpathogenic in humans, they can transduce both dividing and non-dividing cells, and they efficiently transduce some important cell and tissue types. The AAV genome contains three open reading frames, which encode the nonstructural proteins needed for viral replication and virus assembly (rep and aap) and the three structural proteins that assemble to form a 60-mer viral capsid (cap). To create a gene therapy vector, a therapeutic gene of interest is inserted in place of the viral open reading frames to be packaged during vector production. Despite its considerable promise and emerging clinical success, several challenges impede the broader implementation of AAV gene therapy, including the prevalence of anti-AAV neutralizing antibodies in the human population due to natural exposure to the parent virus, low transduction of a number of therapeutically relevant cell types, and an inability to overcome physical transport barriers in the body. These challenges arise since the demands we place on AAV vectors are often different from or even at odds with the properties nature bestowed on their parent viruses. Viral directed evolution - the iterative generation of large, diverse libraries of viral mutants and selection for variants with specific properties of interest - offers a promising means to address these problems. Directed evolution is a high-throughput, molecular engineering approach that our group has adapted and implemented to create AAV variants with novel properties, such as altered receptor binding, altered cell transduction, and altered tissue transduction in the body. In general, the method emulates the process of natural evolution, in which repeated genetic diversification and selection enable the accumulation of key mutations or genetic modifications that progressively improve a molecule's function, even without knowledge of the underlying mechanistic basis for the problem. For AAV, this process has involved mutating wild-type AAV cap genes to create large genetic libraries, which can be packaged to generate libraries of viral particles, each of which is composed of a variant capsid surrounding a viral genome encoding that capsid. A selective pressure - such as the ability to selectively infect HIV-infected T-cells - is then applied to promote the emergence of variants able to surmount these barriers. After each such selection step, the successful variants can be recovered and used as the starting material for the next selection step to further enrich for improved variants. After several such selection steps, the resulting cap gene pool is subjected to additional mutagenesis and selection. After several rounds of mutagenesis and selection, the resulting variants can be analyzed individually for the desired property. I have tried to improve the efficiency of directed evolution of AAV, to create new novel types of AAV libraries, and to utilize these libraries for novel functionality. A thermostable AAV was engineered to compensate beneficial mutations that destabilize the viral capsid, however, there was a trade-off between high and low temperature stability. From these studies, it was discovered that there is variability in the stabilities of natural AAV serotypes that could be exploited to study the mechanism of improved capsid stability as well as compensating destabilizing mutational effects. Alternatively, by allowing AAV to evolve along a neutral network, accumulating mutations that do not have a detrimental effect to natural AAV function but could allow new promiscuous functions, allowed the production of a small but highly useful AAV library. A novel, highly diverse library based on ancestral reconstruction was created with variants inaccessible by standard mutagenesis-based approaches. Finally, utilizing directed evolution of AAV, viral variants were isolated that could selectively infect HIV-infected T-cells for potential use in gene therapy treatments against HIV