Directed Evolution of AAV Serotype 5 for Increased Hepatocyte Transduction and Retained Low Humoral Seroreactivity

Most recombinant adeno-associated virus (AAV) capsids utilized in liver gene therapy have significant levels of pre-existing neutralizing antibodies in the human population. These neutralizing factors limit the patient pools eligible for receiving AAV-mediated therapies. AAV serotype 5 (AAV5) does not face the same barrier of humoral immunity as most AAV serotypes due to its low seroprevalence. However, AAV5 can only facilitate a low level of transgene expression in the liver, constraining its application to a small number of liver diseases. To improve the liver transduction of AAV5 while retaining its low seroprevalence, we constructed a library of AAV5 mutants via random mutagenesis and screened in Huh7 cells. Two molecularly evolved AAV5 variants, MV50 and MV53, demonstrated significantly increased transduction efficiency in Huh7 cells (similar to 12x) and primary human hepatocytes (similar to 10x). All variants had retained low seroreactivity toward pooled human immunoglobulin G (IgG) when compared to AAV5, which was significantly less seroreactive than AAV9. Functional characterization of the mutants also revealed insights into the functions of various domains, especially the VR-I, in the AAV5 capsid. The result is AAV5 variant capsids with much enhanced human hepatocyte transduction, potentially useful for liver-directed gene therapy.