Structural, Viral, and Host Factors of Adeno-Associated Virus Assembly

Abstract

The adeno-associated virus (AAV) is a non-pathogenic virus that has been repurposed as a potent in vitro and in vivo gene delivery vector. Challenges for gene therapy include producing sufficient vector quantities to meet patient need in a cost-effective manner, and the ability to tailor gene delivery specifically to target tissues while minimizing immunological responses or other adverse effects. The AAV capsid is the interface between the host and the genetic cargo, and its protein sequence and structure are the main determinants of vector phenotypes such as tissue tropism, immunogenicity, and production titer. Elucidating capsid assembly mechanisms and the structural determinants of efficient assembly can directly inform vector production and capsid engineering strategies aimed to overcome current limitations for gene therapy. This dissertation describes a method employing a synthetic viral toolkit to identify capsid structural determinants of a queried AAV phenotype. As proof-of-principle, first we identify an inter-monomeric capsid motif that determines dependence on the viral cofactor the Assembly-Activating Protein (AAP) for capsid assembly, and next the site of interaction between AAP and the capsid proteins. Biochemical and molecular biology approaches reveal how residues on the capsid and on AAP can be modulated to disrupt or enhance assembly. In turn, these studies reveal previously undescribed functions of AAP, as well as host responses to the intrusion of virion assembly. Finally, unbiased genetic and proteomic approaches are used to identify several potential cellular assembly and restriction factors. Initial functional validation of these factors suggests critical roles for Hsc/Hsp70 (and a specific subset of its cofactors), the vacuolar-specific H+ ATPase, and cyclin-dependent Kinase 2 in vector production. We aim to apply our findings to establish cell lines and/or culture conditions to improve upon current vector production systems. Taken together, these studies on assembly from the perspectives of both virus and host provide multifaceted insight into AAV biology, and can aide rational vector design tailored to specific disease indications while preserving, or enhancing, the primary requirement of any vector: its assembly.