294. Characterizing AAV CaptureSelect Affinity Ligand Interactions

Adeno-associated virus (AAV) is one of the most widely studied viral vector systems for therapeutic gene delivery. This application has already experienced success in several human clinical trials, including the treatment of hemophilia B with an rAAV8 vector expressing therapeutic levels of Factor IX protein and for the treatment of Pompe disease, utilizing AAV1 vectors. Recently, an rAAV1 vector packaging a gene for the treatment of lipoprotein lipase deficiency was approved as the first gene therapy drug, validating the utility of this system. However, the effective production and purification of enough viral vector for use in research, preclinical studies, and human treatment is essential for success. Towards this goal, camelid single domain antibody fragments directed against AAV were used to make affinity resin products suitable for fast, one-step purification of AAV vectors of several serotypes. This affinity based purification method can replace current standards of density purification, which are time consuming and require large volumes of reagents, such as sucrose or iodixanol. At present, the collection of affinity resins suitable for AAV purification, i.e. AVB-Sepharose™, POROS™ CaptureSelect™ AAV8 and -AAV9, show varied affinity for different AAV serotypes, resulting in varied efficacies of virus purification. To better understand these differences and improve broad range utility, it is important to understand how these affinity ligands bind to the surface of the AAV capsids. We have thus mapped their binding footprints on several AAV serotypes, including AAV1, AAV5, and AAV9, using cryo-electron microscopy and image reconstruction combined with pseudo-atomic modeling. The sites for different affinity ligands are clustered on regions of the capsid that are common among most serotypes, but display minor variations which likely account for the varied affinities. This information will assist in the synergistic development of affinity ligands with broader serotype coverage and AAV vectors with improved purification outcomes.