Approach for in vivo gene delivery into hematopoietic stem cells for hemophilia A therapy

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ABSTRACT: We will test a new approach for the production of therapeutic proteins secreted from blood cells after in vivo gene delivery into hematopoietic stem cells (HSCs). This approach involves the mobilization of HSCs from the bone marrow followed by a single intravenous injection of integrating helper-dependent HDAd5/35++ adenovirus vectors. HSCs transduced in the peripheral blood return to the bone marrow where they persist long-term. Transgene integration is achieved either in a random pattern using a transposase or, in a site- specific pattern, through homology-directed DNA repair mechanisms. For a secreted transgene product, we will focus on human factor VIII expressed in erythrocytes after in vivo factor VIII gene transfer into HSCs. In contrast to currently used rAAV-mediated liver-directed hemophilia gene therapy, our technically simple and cost-efficient approach has the potential for a life-long cure with induction of tolerance to factor VIII. The Specific Aims are 1. Increase the efficacy and safety of transposase-based HDAd5/35++ in vivo HSC transduction through optimization of mobilization and vector injection regimens and through HSC in vivo expansion or selection mechanisms. 2. Test new HDAd5/35++ vector systems for targeted integration, including a vector that carries both a CRISPR-Cas9 to create site-specific DNA breaks and the homology template for integration. 3. Test the best in vivo HSC transduction system in a mouse model for hemophilia A. 4. Perform a pilot safety and efficacy study in non-human primates, which are the most adequate model for potential future studies in humans. The proposed 6-month study with repeated blood and bone marrow sampling will allow us to predict potential long-term side effects on hematopoiesis and follow the expansion of gene-edited HSCs over time.