Metabolic biotinylation provides a unique platform for the purification and targeting of multiple AAV vector serotypes

Abstract

The development of rationally designed targeted gene delivery vectors is an important focus for gene therapy. While genetic modification of AAV can produce vectors with modified tropism, incorporation of targeting peptides into the structural context of the AAV virion often results in loss of function or loss of virion integrity. To address this issue, we have developed a targeting system using metabolically biotinylated AAV. We generated serotype 1, 2, 3, 4, and 5 AAV capsids with small peptide insertions that are metabolically biotinylated in packaging cells during vector production by coexpression of the Escherichia coli BirA, biotin ligase, gene. Biotin moieties are exposed on the surface of assembled AAV particles and can interact with avidin. Metabolically biotinylated AAV vectors produced in this manner maintained endogenous titer and tissue tropism, could be purified on monomeric avidin resin, and could be retargeted to cells engineered to express an artificial avidin-biotin receptor. This technology provides not only a single platform for the purification of multiple AAV vector serotypes, but also a means for the development of multiple targeted AAV vectors utilizing a single capsid modification via straightforward avidin-biotin ligand coupling.