Novel High-Throughput Methods to Study AAV Immunity Using Combinatorial Capsid Libraries

Abstract

Adeno-associated virus (AAV) is a helper-dependent, nonpathogenic virus that has shown significant promise as a gene therapy vector. As a result of abundant natural AAV infections, a majority of the population harbors neutralizing antibodies against the capsid, potentially confounding transduction of therapeutic vectors. Researchers have made numerous efforts to engineer AAV vectors to evade humoral immunity, but incomplete understanding of the pre-existing antibodies in humans has hampered these attempts. In this work, I aim to explore these questions through AncAAVs, a series of capsids previously designed in our lab through ancestral sequence reconstruction. First, we determine optimal conditions for AAV library production to minimize noise generated by cross-packaging and mosaicism of different capsid subunits. Through a series of novel assays with representative libraries, we find that 10- to 100-fold reduction in library DNA input results in minimal detectable cross-packaging. Next, we will use those insights to produce and characterize the Anc80 viral library with regards to production, in vitro transduction, and murine liver tropism. We identify a single variant position in the Anc80 library which mediates liver transduction ability without strongly affecting transgene expression in muscle or heart, offering potential strategies for the creation of liver de-targeting vectors. Finally, we make use of the entire set of AncAAV libraries to identify capsid residues associated with neutralization, first by monoclonal mouse antibodies and then by human polyclonal sera. These studies together demonstrate the power of library approaches to uncover new insights into AAV capsid biology, which hopefully will guide the design of the next generation of gene therapy vectors.