Investigators' Abstract) The ultimate goal of HIV-1 directed gene therapy is to inhibit virus replication and the development of AIDS. Clinical studies have focused on human cell and gene transfer for this purpose, and studies have revealed both a low frequency of marked cells and a very low level of gene expression. Thus, several essential issues will need to be addressed before gene therapy will become a treatment reality, particularly for HIV-1 infected children. As an alternative to ex vivo transduction of hematopoietic stem cells (HSC), these investigators propose to investigate the in vivo transduction of HSC in their native environment, the milieu of the bone marrow. Studies in uninfected rhesus neonates will initially be performed to determine the requirements for efficient gene transfer to the HSC. The investigators will explore the most effective vector system (Moloney murine leukemia virus {MLV} versus HIV-1-derived lentivirus), the effects of cytokines on the in vivo transduction efficiency of HSC, and the number of intramarrow infusions necessary for efficient marking of HSC and their progeny (Specific Aim 1). Newborns will be transferred intramarrow with either MLV/VSV-G or HIV/VSV-G each marked with the enhanced green fluorescent protein (EGFP) as a reporter gene. Blood and marrow will be collected 2 weeks then monthly post-transfer for 12 months. Samples will be assessed for transduction and expression (flow cytometry, PCR, hematopoietic progenitor assays). While it is clear that juvenile/adult animals generate a potent immune response to retrovirus vectors and their transgenes, the immune consequences of the proposed studies are uncertain due to the age of the recipients, and will be investigated (Western blots, antibody titers, lymphocyte proliferation assays. The investigators will then utilize the best in vivo transduction approach developed in Specific Aim 1 to deliver an antisense Tat/Rev gene cassette to the HSC of SIV-infected rhesus neonates (Specific Aim 2). Siv-infected newborns on antiretroviral chemotherapy (PMPA) will receive intramarrow transfers, and blood and marrow collected 2 weeks than monthly post-transfer for 12 months. The presence of the vector transduced cells in samples will be detected by Southern blot of DNA-PCR, and expression measured by Northern blot, RT-PCR, or FACS analysis. Plasma- and PBMC-associated veremia, and levels of SIV in plasma will be quantitated (bDNA assay). Studies will focus on the generation of anti-SIV gene-marked HSC and mature lymphohematopoietic cells, and the expression of the antiviral elements(s) in these cells. Thus, the goal of the investigators is to explore a safe and efficacious approach for pediatric gene therapy of HIV-1 infection targeting HSC using in vivo transduction for the delivery of antiviral genes. These studies are directly relevant to infants and adults infected with HIV-1.
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