Improving the reach and efficacy of gene therapy by in vivo EV targeting

The goal of this project is to extend the reach and efficacy of adeno-associated virus (AAV)-based gene therapy by enlisting endogenously produced extracellular vesicles (EVs) to transport engineered transgene mRNA or protein products among cells and across tissues. The motivation for this project is that many inherited disorders are refractory to current AAV-based gene therapy for 1 of 2 reasons. First, the fraction of cells transduced by AAV when administered systemically at a well-tolerated dose is limited and for some disorders may be insufficient to rescue phenotype. Second, even if initial transduction efficiency is high, because AAV is non-replicating and predominantly non-integrating, viral genomes tend to be lost quickly from cells in high mitotic tissues by simple dilution due to repeated cell divisions as the child grows. Of note, booster shots are not a good option because of neutralizing antibodies elicited by the initial dose. We propose a conceptually simple solution to these problems: targeting intracellular transgene product – either as protein or as mRNA – to endogenous EVs so that cells containing viral DNA will be able to share their expressed transgene product locally with neighboring cells and systemically via transport through the bloodstream, including across the blood- brain barrier. Specifically, we hypothesize that EV-facilitated transfer of transgene-encoded protein and/or mRNA will increase the percentage of transgene product-positive cells in key tissues and will allow cells in low mitotic tissues that retain viral DNA to share their expressed transgene product with cells in other tissues. Here, we propose to test this hypothesis using an existing rat model of classic galactosemia administered scAAV9 virus encoding human galactose-1-P uridylyltransferase (GALT) either untagged or carrying 1 of 3 different EV- targeting tags. The results of the proposed 2-year study will inform the direction of future research and set the stage for application of this approach to gene therapy options for a large number of genetic disorders for which there is currently no effective treatment. 1