The goal of these studies is to identify the principle mechanisms that control the transcriptional status of genes introduced into long-term self-renewing, hematopoietic stem cells (LT-HSC) using retroviral vectors. Understanding transcriptional silencing may lead to the design of retroviral vectors that stably express molecular therapeutics in hematopoietic cells independent of the site of retroviral integration. Murine retroviral vectors have been the most widely used vehicles for gene delivery into HSC in both mice and larger vertebrate organisms including man. The success of approaches used to date has been hampered by a number of variables including a poor understanding of stem cell biology in larger mammals, low transduction efficiencies of primate HSC, and the lack of sustained gene expression from viral vectors. Clinical results have led to the recognition that more basic science research needs to be done in each of these areas if gene therapy is to be successful in humans (Orkin and Motulski report to the NIH; December, 1995). One of the most challenging hurdles which has received relatively little attention is the mechanism(s) that cause rapid transcriptional inactivation of the majority of retroviral integrants. Understanding how genes are silenced and, perhaps even more importantly, why certain proviral integrants remain transcriptionally active, is critical if retroviral vectors are to be used effectively in treating congenital and acquired hematopoietic deficiencies. With this goal in view, these studies propose the following: (1) to determine whether transcriptional inactivation of retrovirally-expressed genes occurs downstream of LT- HSC; (2) to examine the influence of methylation and chromatin structure in transcriptional silencing; (3) to ascertain whether inactivated retroviruses that originally expressed the green fluorescent protein (GFP) can be reactivated for expression in vivo using chromatin, or methylation-modifying drugs like sodium butyrate, trichostatin A or 5- azacytidine; (4) to clone dominant, cis-acting sequences in the genome that insulate certain proviral integrants from transcriptional inactivation; (5) to use a dual fluorochrome reporter assay and locus control regions to engineer vectors that are not subject to transcriptional inactivation or position effect in hematopoietic cells. When these basic science issues have been addressed in the mouse, both mouse (Aim 5) and large animal model systems will then be used to apply the fundamental principles learned in these experiments to clinically important disease models.
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| Cotta, Claudiu V; Zhang, Zheng; Kim, Hyung-Gyoon et al. (2003) Pax5 determines B- versus T-cell fate and does not block early myeloid-lineage development. Blood 101:4342-6 |
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