The NHGRI intramural program has expanded research efforts to integrate virus vectors into adult hematopoietc stem cells (HSC) to combat HIV infection. The HSC is the ultimate progenitor of all peripheral blood cells, including the CD4+ lymphocytes that are infected by HIV 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 two novel retroviruses are plentiful on HSC, unlike the conventional retrovirus receptors, which are not present on HSC. We have demonstrated high levels of gene transfer into human HSC using retrovirus vectors with these new envelopes. In the coming year, using mouse and sheep xenograft models. We will extend these studies to include marrow from patients, and will evaluate transduction with both marker and therapeutic vectors. 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. Because of new safety concerns regarding insertional leukemogenesis in a gene therapy trial, NHGRI intramural researchers have compared the integration sites of lentivirus and oncoretrovirus vectors. lentivirius vectors integrate in transcribed regions while oncoretroviruses integrate into the regulatory regions of genes. We have also demonstrated that the protein Hmgb3 plays a critical role in hematopoietic cell cycle progression. in the future we plan to locate the target genes for Hmgb3 and to test whether stem cell cycling can be manipulated through this pathway. 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. We have demonstrated that an insulator from the chicken beta globin locus can suppress silencing in vectors and are evaluating two new insulators that we have characterized from the ankyrin gene locus. these insulators are more compact that the chicken insulator and should allow higher titer vector preps to be developed. 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 has initiated 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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