Structure-Guided Rational Design of Adeno-Associated Viral Capsids with Expanded Sizes

The application of recombinant Adeno-Associated Virus (rAAV) vectors as gene delivery vehicles has been limited by the modest packaging capacity (5.2kb) of rAAV. Th is limitation is imposed by the capsid size of 25nm diameter, which is determined by the capsid’s T=1 icosahedral geometry. Current methods for rAAV delivery of oversized cargo, typically involving co-delivery of dual vectors, suffer from challenges such as low efficiency in co-infection and reassembly of full-length products, indefinite ratios of the two vectors in individual cells, and necessity of re-design and re-validation for every new cargo. Inspired by the size polymorphism observed in natural viruses, we hypothesized that the T=1 icosahedral geometry of AAV capsids could be changed so that each capsid is built from more than 60 subunits. Here we present two rational design strategies that lead to eXtra Large AAV capsids (XL-AAVs). Th ese strategies involve modifying the intersubunit interactions and the assembly pathway of AAV capsids. Capsids engineered through both strategies form heterogeneous, 35nm-70nm spherical particles (Figure 1). Th e XL-AAV capsids can still package rAAV genomes, and the same protein design principles can be applied across multiple serotypes we tested (including AAV9, AAV2, AAV5, and AAVDJ). Ongoing work seeks to determine whether the XL-AAV capsids can fully protect and deliver conventionally oversized rAAV genomes. These design principles and the emerging XL-AAV capsids represent the first steps towards creating rAAV vectors with larger clone capacities, opening a path towards the delivery of disease-related genes and genetically-encoded tools with long coding sequences in single rAAV vectors