| Viral infections generally begin with virus-glycoconjugate interactions on the host cell surface. Adeno-associated Virus (AAV) serotypes have been shown to bind to sialylated glycans and heparan sulfates for cellular attachment. However, the underlying molecular mechanism and its impact on AAV tropism in hosts have not been well understood. In this dissertation, terminal N-linked galactose was identified as the primary receptor for AAV serotype 9 (AAV9). Moreover, recombinant sialidase was explored as an adjuvant to dramatically enhance AAV9 gene transfer both in vitro and in vivo. More in-depth analysis of the effects of sialidase on the transduction, tropism, and pharmacokinetics profiles of AAV9 indicates that higher binding avidity between AAV9 and galactose alters its tropism, shifting it from a systemic towards a hepatic phenotype. Using AAV9 as a model system, the functions of glycan receptors in determining AAV transduction efficiency in vitro and its tropism in vivo were elucidated. In addition, it was also showed that the blood circulation kinetics of AAV vectors are tightly regulated by their binding avidities with cognate glycan receptors on tissues and blood components. In parallel, key amino acids in the galactose binding footprint on the AAV9 capsid were identified by screening of a random mutagenesis library of AAV9. Furthermore, grafting of the galactose binding footprint from AAV9 onto other AAV strains yields a series of novel AAV variants with enhanced transduction efficiency, as well as a rapid on-set of transgene. This work also demonstrates the orthogonality and modularity of glycan binding motifs from several naturally occurring AAV serotypes, as well as the adaptability of the AAV capsid for the incorporation of new functionalities. By establishing the link between the first step of AAV infection and its tissue targeting profiles, a novel strategy was proposed to design and construct AAV vectors with organ-targeting or detargeting phenotypes. In conclusion, this dissertation sheds light on the crucial roles of glycan receptors in AAV infectious pathways, and paves a new avenue for vector development for gene therapy applications. |
