The NHGRI intramural program will be expanding research efforts in integrating adult hematopoietc stem cells (HSC) into virus vectors in the effort to combat HIV infection. The HSC is the ultimate progenitor of all peripheral blood cells, including the CD4+ lymphocytes that HIV infects and the macrophages that can sequester HIV for long periods of time in tissues. If an anti-HIV element can be introduced into HSC, it will be passed along to all of the progeny of that HSC, ensuring the continuous, life-long production of HIV resistant cells. If this therapy were completely successful, this treatment would permanently protect the patient from HIV spread and no further treatments would be required. The successful modification of HSC by viral vectors has three important steps. First the viral vector must bind to a specific receptor on the surface of the HSC. HSC are a rare population of cell in the bone marrow, accounting for less than 1/10th of 1% of all bone marrow cells. Researchers in the NHGRI intramural program have developed methods to purify human HSC. We have determined that the receptors for the retroviral gene transfer vectors currently in use, while plentiful on mature CD4+ lymphocytes, are not present on HSC. Thus HSC are resistant to transduction by these vectors. NHGRI intramural researchers have begun an analysis of virus receptor levels on highly purified human HSC and have determined that several novel virus envelopes have receptors present at high levels on HSC. In the coming year, the transduction of human HSC with these novel viruses will be compared and analyzed using mouse and sheep xenograft models. We expect that these studies will identify the optimal virus envelope to maximize binding to HSC, leading to improved introduction of anti-HIV elements. The second important step in the modification of HSC requires the HSC to divide so that the new anti-HIV sequences can become integrated into the DNA of the target cell. Normally, HSC divide infrequently, while their progeny proliferate rapidly. This allows a small number of stem cells to provide millions of red and white blood cells to the circulation each day. HSC division can be manipulated by the use of hematopoietic growth factors. NHGRI intramural researchers plan to use highly purified human HSC to determine the optimum combination of growth factors that will promote HSC division during the exposure to virus vectors, while preserving the ability of the HSC to generate a life-time supply of red and white blood cells. Initially, these studies will take place in cell cultures, after which these methods will be tested in the mouse and sheep xenograft studies. We expect to combine the optimal combination of hematopoietic growth factors to promote HSC division with the optimal virus envelope to develop a highly efficient method to introduce anti-HIV elements into HSC. The final important step for successful modification of HSC with anti-HIV elements is that the anti-HIV elements have to be produced in the mature progeny of the HSC at all times so that the cells are always prepared to interrupt HIV infection. Many groups have shown that viral vectors can become ?silenced? over time and stop making the critical elements. To combat silencing, NHGRI intramural investigators are developing and evaluating different virus vectors that contain genetic elements to prevent silencing. Among the vectors under development are vectors containing ?insulator elements? that prevent the type of changes associated with gene silencing. Alternatively, vectors with some of the HIV virus regulatory elements (but not the HIV genes) are being evaluated as it is hypothesized that HIV itself is resistant to silencing in cells. NHGRI intramural investigators will evaluate the long term production of anti-HIV elements in cells in the mouse and sheep xenograft models to determine the optimal virus vector to express the anti-HIV elements in blood cells, particularly CD4+ lymophocytes. The combination of these three initiatives should significantly improve the prospects for successful HSC based AIDS gene therapy. It is anticipated that the results of these studies can be combined into a clinical trial for AIDS gene therapy in the near future. NHGRI will initiate a genetic approach to AIDS that takes full advantage of the strengths of the intramural NHGRI clinical research program. These approaches are aimed at understanding the genetic component of innate HIV resistance. NHGRI clinical researcher will recruit and evaluate families in which HIV positive individuals without symptoms of AIDS have been identified. The families will be screened for changes in the coding sequences for the HIV co-receptor proteins and their blood cells will be tested in vitro to see if their cells are susceptible to HIV. If specific changes in the co-receptor molecules can be shown to be associated with inherited resistance HIV infection, these molecules would become prime candidates for either gene therapy or molecular decoy strategies to combat AIDS. As larger numbers of families are recruited, the NHGRI Inherited Disease Research Branch will use powerful gene mapping and statistical genetics analyses to identify additional genetic loci that contribute to HIV resistance. It is anticipated that the results of these initiatives will provide novel ideas to evaluate for the treatment and prevention of AIDS. The NHGRI Genetic Counseling program will initiate an effort to counsel and educate individuals with AIDS. The Genetic Counseling program has completed an extensive study of the most effective counseling techniques for families with inherited immune disorders. It is of great interest to learn in what ways the attitudes and behavior of individuals and their relatives are influenced by whether an immune disorder they may have is inherited or acquired. The NHGRI AIDS initiative will compare counseling approaches that have been shown to be effective in inherited immunodeficiency diseases with counseling for AIDS families. Such psychological studies are expected to reveal whether different approaches are necessary to help families cope with AIDS and its consequences.
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