256. A Novel Rationally Designed AAV Capsid Yields a Potent Neurotropic Gene Therapy Vector

Recent clinical trials have demonstrated safety and efficacy of adeno-associated virus (AAV)-mediated gene therapy, and precipitated the approval of the first gene therapy product for commercial use. Although AAV is the most commonly used vector, there are limitations due to its restricted biological activity. This can be partially overcome by aligning capsid choice and target tissue. Tailoring of AAV capsids has traditionally been accomplished by simply selecting the most suitable available serotype or by further optimization of available serotypes by peptide insertion or capsid reshuffling and in vivo selection in mouse or humanised mouse models. A novel approach based on in silico reconstruction of ancestral viruses recently yielded a promising vector for gene therapy of diseases that affect liver, muscle or retina. Here we report an alternative evolutionary approach to AAV capsid design based on the introduction of amino acids that have been found at the other end of the evolutionary spectrum. Specifically, we have designed a novel AAV2 capsid by including amino acids that are conserved in natural AAV isolates found in human pediatric tissues. We have determined the structural and immunological characteristics of this novel AAV2-based capsid and performed biodistribution studies. When the bioactivity of the vector was evaluated in various in vivo contexts the data revealed extraordinary transduction characteristics in eye and brain tissues. Intracranial injections in mice and rats revealed a significantly increased spread and enhanced transduction of brain tissues as compared to AAV2, with evidence for retrograde transport. Additionally, intra-ocular injections in adult mice revealed a marked enhancement of transduction of photoreceptor cells by the novel vector when compared to AAV2. Peripheral injections also demonstrated strong affinity for the brain and showed little evidence for transduction of non-neuronal tissues. Importantly, we were able to demonstrate superior performance in a disease model that affects the central nervous system when compared to other serotypes that have a strong affinity for neuronal tissues. In summary, we have engineered a novel capsid to include key residues found in natural variants of AAV2, which yields a unique gene therapy vector for the treatment of diseases that affect the central nervous system.