In vivo hematopoietic stem cell genetic modification by viral particles - PROJECT SUMMARY Hematopoietic stem cell (HSC) gene therapies with lentiviral or CRISPR based gene modification are demonstrating favorable safety profiles and remarkable efficacy to treat severe monogenic blood disorders with unmet clinical need, including X-linked Severe Combined Immunodeficiency (X-SCID) and Sickle Cell Disease (SCD), with feasibility of rapid progress from target discovery and preclinical validation to first-in-human trials. Despite these successes, the technologies and procedures used in these trials require complex ex vivo bespoke manufacturing of autologous cell products, are expensive and difficult to scale to treat many patients, and intrinsically associated with significant risks related to ex vivo manipulation, myeloablative chemotherapy and bone marrow transplantation. We here propose a novel technology platform based on alpha-retroviral vectors that shares many of the advantages with the currently dominating delivery platform, lentiviral vectors, but has increased flexibility to deliver diverse therapeutic payloads, including non-integrating Cas9-based genome editors, high potential for in vivo gene delivery, and compatibility with scalable production systems. The long- term goal of our proposal is to further develop this platform for the delivery of gene therapy payloads (integrating DNA and Cas9/base editor ribonucleoprotein complexes into HSCs in vivo to enable simple, more economical and widespread application of gene therapies to serve diverse patient populations. A shorter term goal is to employ the same technology for improved ex vivo delivery of gene therapy payloads to HSCs. Our central hypothesis is that a single platform based on alpha-retroviral vectors is suitable for ex vivo and in vivo delivery of genetic therapies with diverse requirements in terms of the type of therapeutic payload and targeting specificity. In preliminary studies, we have demonstrated that both integrating vectors and non-integrating genome editors can be delivered into HSC ex vivo and in vivo. Here, we will capitalize on these results and further expand the reach of these innovative genetic engineering tools with the objectives to: i) develop integrating vectors to deliver a curative transgene to HSCs in vivo as a modality to treat X-SCID; ii) engineer virus-like particles for the delivery of Cas9 into quiescent HSC with minimal toxicity both ex vivo and in vivo to treat SCD; and iii) to modify virus-like particles for the transfer of base editors into HSCs for correction of the common mutation underlying the bone marrow failure disorder Shwachman-Diamond Syndrome. The alpha- retroviral based viral particle technology will be used to develop treatments for this exemplary group of diseases and is also intended to address key general shortcomings of current GT approaches and could be readily transferable to many other monogenic diseases.
