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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054766-02
Application #
6150649
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Badman, David G
Project Start
1999-03-01
Project End
2003-01-31
Budget Start
2000-02-01
Budget End
2001-01-31
Support Year
2
Fiscal Year
2000
Total Cost
$212,644
Indirect Cost
Name
University of Alabama Birmingham
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
004514360
City
Birmingham
State
AL
Country
United States
Zip Code
35294
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Swindle, C Scott; Kim, Hyung G; Klug, Christopher A (2004) Mutation of CpGs in the murine stem cell virus retroviral vector long terminal repeat represses silencing in embryonic stem cells. J Biol Chem 279:34-41
Purohit, Sheetal J; Stephan, Robert P; Kim, Hyung-Gyoon et al. (2003) Determination of lymphoid cell fate is dependent on the expression status of the IL-7 receptor. EMBO J 22:5511-21
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
Klug, C A; Cheshier, S; Weissman, I L (2000) Inactivation of a GFP retrovirus occurs at multiple levels in long-term repopulating stem cells and their differentiated progeny. Blood 96:894-901