(310a) Molecular Engineering of Adeno-Associated Viral Vectors for Targeted Gene Delivery

Rational protein engineering efforts to mediate targeted gene delivery to a defined cell type of interest have met with limited success, likely due to the complex nature of virus-cell interactions. In such situations, a broader ?black box? approach to engineer efficient viral vectors through random diversification and high-throughput selection can still succeed as evidenced by the recent creation of novel viral gene delivery vectors with customized molecular properties, such as enhanced resistance to neutralizing antibodies.

Due to the natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, efficient gene delivery within the central nervous system (CNS) has mainly occurred to neurons. Astrocytes possess several attributes of strong clinical importance within the CNS such as maintaining normal CNS homeostasis and as a causative agent in a host of genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, but efforts to develop more efficient gene delivery vectors for astrocytes and other glial cells have met with limited success.

Here, we sought to evolve a highly efficient AAV vector capable of improved astrocyte gene delivery by employing directed evolution with an assorted panel of innovative AAV libraries, constructed using random mutagenesis, DNA shuffling, random peptide display, and a new semi-random peptide replacement strategy. This novel peptide replacement strategy exploits the inherent modularity and structural plasticity of the AAV capsid loops through bioinformatics-based design of new AAV capsid loops based upon analysis of over 130 AAV cap genes. Multiple evolutionary cycles of diversification followed by selection on primary human astrocytes resulted in the creation of several enhanced AAV vectors. These novel variants mediate gene delivery to both human and rat astrocytes in vitro up to 15-fold more efficiently than the parental AAV serotypes, and injection into the adult rat striatum led to astrocyte gene delivery levels up to 16% of the total transduced cell population, compared to very low levels of gene delivery by the parent serotypes. Furthermore, several of these variants mediate efficient gene delivery to Muller glial cells upon injection into the retina of rats, whereas no transduction was observed by the parent serotypes, further highlighting the general utility of these variants for efficient glial transduction. These novel highly efficient AAV vectors will greatly extend the use of AAV vectors within the CNS and disease models by enabling transduction of astrocytes and other glial cell types. Furthermore, validation and analysis of these novel AAV libraries will improve molecular engineering efforts to generate customized viral vector with defined gene delivery properties.