266. G-Force Loading of Virus Vectors into Vesicles for Enhanced Gene Therapy Vehicles

Abstract

Adeno-associated virus (AAV) vectors have shown remarkable efficiency in a number of preclinical models of disease in several organs including the eye, brain, muscle, heart, and liver. A major limitation to long term transduction, especially via the systemic route, is pre-existing immunity (humoral and cell-mediated) as well as the high vector load required to achieve sufficient levels of gene expression in the desired organ. Natural nanoparticles released from all cells, called extracellular vesicles (EVs), may have utility in creating better AAV vectors for human gene therapy. We have previously shown that harvesting AAV vectors from the media of 293T producer cells contains AAV vectors endogenously associated with EVs (called ev-AAV, aka vexosomes). We have gone on to demonstrate that 293T-derived ev-AAV can evade neutralizing anti-AAV antibodies and greatly increase transduction in mice. An alternative strategy would be to load purified AAV vectors into separately purified EVs. This would have the benefit of using EVs from a variety of cellular sources with desired biological activity and also to use donor-derived autologous EVs. Here we demonstrate a simple method for loading EVs with AAV vectors using high speed centrifugation forces.Iodixanol density gradient-purified AAV was mixed with conditioned media containing EVs from 293T cells and also primary human peripheral blood mononuclear cells (PBMCs) and then ultracentrifuged to co-pellet EVs and AAV. The pellet was resuspended in media and used in transduction assays and anti-AAV antibody neutralization assays.Strikingly we found that this process bestowed greatly enhanced transduction of cells in culture (36-fold) as well as resistance to neutralizing anti-AAV antibodies. To demonstrate that EVs were essential to this enhancement, we mixed AAV with plain media (no EVs) or depleted conditioned media of EVs before addition of AAV using either Triton-X-100 EV lysis or ultracentrifugation. In all cases there was a large decrease in the transduction efficiency and antibody evasion compared to conditioned EV media mixed with AAV.Together these data suggest that g-force loading of AAV into EVs represents a promising method to increase performance of the vector. We will continue to optimize the protocol and test the gene delivery functions in vivo.