Targeted in vivo gene transfer to hematopoietic stem cells

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DESCRIPTION (provided by applicant): Efficient gene transfer to human hematopoietic stem cells (HSC) has the potential to correct a number of human hematologic and non-hematologic diseases. At present, gene transfer into HSC's is typically achieved ex vivo, involving isolation of stem cells, transferring a corrective gene into the cells and re-infusion of cells into the patient. For the purpose of achieving therapeutic gene transfer, there would be great utility in a vector that could achieve targeted gene transfer in vivo to HSC. For this R21 proposal we aim to develop and test in vivo lentiviral based gene transfer vectors that will target infection through molecules that are expressed on the surface of hematopoietic stem cells. We will (i) develop lentivirus vectors for targeting infection of HSC. This will be achieved by fusing protein domains that bind cell surface receptors expressed on HCS (including CD34, and the kinase receptors c-kit and Flt3) onto a mutant Sindbis virus envelope that is severely impaired for binding its receptor but is competent for membrane fusion. These vectors will be extensively characterized using in vitro cell culture systems. We will then proceed to (ii) evaluate vectors generated for targeting infection of hematopoietic stem cells in vivo. We will investigate two routes of delivery for targeting lentiviral vectors to HSC: (a) injection into the bone marrow and (b) systemic delivery after mobilization of HSC out of the marrow into blood. These experiments will critically test the feasibility of targeting infection to HSC in vivo. Targeting vector to cells in vivo has the potential advantage of increasing gene transfer to cells while decreasing viral load. It also avoids gene transfer to cells that could impede gene therapy i.e. dendritic cells. The targeting of hematopoietic stem cells will provide proof of principle for in vivo targeting and support its use as a therapeutic approach for key hematologic disorders, such as immunodeficiencies, AIDS and Fanconi anemia. Furthermore, results from these studies will have significant implications for targeted therapeutic gene transfer using retroviral vectors in general. Public Health Relevance: This grant proposal aims to develop and test viral vectors that are engineered to infect a specific cell type in the body. Here we propose to develop these to target infection of stem cells that are responsible for making all the cells of the blood. The development of these viral vectors will enable treatment of blood disorders by direct injection of these vectors into the body. The discoveries we make can also be applied to other diseases.