(PROJECT 2- CHEN) The hypothesis to be tested in this project is that transplant of gene-modified HSPC and T-cells give rise to repopulating cells exhibiting normal differentiation. The other projects proposed in this program investigate genetic modifications of HSPC and/or T-cell populations to either resist infection from HIV-1 (Project 4: Symonds), provide anti-HIV-1 effector activity (Project 3: Kitchen), or improve the efficiency of engraftment (Project 1: An). We propose to track behavior of individual transduced cells within the population of transplanted and repopulating cells by using vector integration sites (VIS) as a marker to uniquely identify individual repopulating clones and assess the behavior of these clones for repopulation characteristics. Repopulation of normal non-gene modified HSPC and T-cells results from the additive contributions of hundreds to thousands of stem and progenitor cells which give rise to clones of repopulating mature blood cells. Classicly, it was thought that these repopulating cells are relatively homogeneous in regards to features such as longevity and differentiation, however the recent studies tracking the behavior of individual HSPC clones in mice and our recent clonal tracking studies of HSPC in non-human primates demonstrate that these stem and progenitor cells represent complex mixtures of cells having diverse functional properties. Heterogeneous populations of HSPC differ in regards to multiple factors, including relative contribution to repopulation, self-renewal, commitment to differentiation lineage, and longevity. Gene modification with transgenes which further alter the genetic properties of these cells introduces additional possibilities for inadvertent and potentially adverse behavior changes. Clonal analysis of gene-modified HSPC can address the likelihood of successful HSPC genetic therapy for HIV-1 disease. For example, it is important to have long- term repopulation by many gene-modified HSPC clones (polyclonal) capable of multilineage differentiation, particularly into HIV target cells, myeloid and lymphoid cell, and well represented in all primary and secondary lymphoid tissues. Analysis depends upon having a high-throughput technology that can accurately quantitate and sensitively detect clones. To that end, we have adapted the genomic pyrosequencing technology for this purpose and published a validated high throughput assay which enables a quantitative and sensitive enumeration of individual VIS and, in combination with intensive bioinformatics computation, determines the relative frequencies of progeny derived from individual HSPC within a given tissue sample. This project will utilize humanized mice and non-human primate tissue samples derived from Projects 1 and 3 for clonal tracking.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program--Cooperative Agreements (U19)
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Special Emphasis Panel (ZAI1)
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University of California Los Angeles
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