Delivery of genes encoding short interfering RNA (siRNA) to target human immunodeficiency virus (HIV) may represent an effective approach to interfere with virus infection. Human hematopoietic stem cells (HSCs) represent the ideal target for gene delivery since they can readily be isolated with well-established procedures and can be cultured to allow ex vivo gene delivery. For successful treatment of HIV infection with the gene therapy approach, efficient siRNA expression and high percentage transduction are the two most critical factors. In addition, generating stable packaging cell lines that produce clinically acceptable HIV vector stocks would facilitate not only clinical studies but also preclinical experiments in large animals. In this proposal, we will evaluate various vector design and cis elements to optimize siRNA expression in HSCs. We will study the design of HIV vectors for efficient siRNA expression. We will evaluate different pol Ill promoters and shRNA gene dosage on the anti-HIV activity in human T cells and in myelomonocytic cells differentiated from CD34+ cells in culture. We will evaluate different cis-acting DNA elements for their ability to stabilize siRNA expression. To increase the fraction of the transduced cells, we will evaluate the potential of expressing methylguanine methyltransferase (MGMT) or type II inosine-5' monophosphate dehydrogenase (IMPDH2) to enrich and expand the transduced HSCs in vivo. HIV vectors containing the selectable marker will be used to transduce HSCs, followed by cell injection into the SCID-hu mouse model. Upon in vivo drug selection, the engraftment, expansion and resistance to HIV-1 infection of the differentiated T cells will be analyzed. To establish stable packaging cell lines for large-scale vector production, we will test a Cre-loxP-based system for inducible expression for HIV Gag/Pol and the G protein of vesicular stomatitis virus. The cell lines will be tested for their ability to generate high-titer vector stocks under the inducible condition.
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