This project is focused on optimizing lentiviral vector mediated gene transfer into long-term repopulating cells, on the evaluation of globin gene vectors, on developing strategies to activate the endogenous globin genes and on evaluating the safety of globin gene therapy approaches. The proposed research involves stem cell targeted gene transfer into autologous cells which will be done in a rhesus transplantation model whereas strategies to optimize globin vectors and to evaluate their safety will be performed using cultured erythroid cells, cell lines and murine models in addition to the rhesus model. We have developed a lentiviral vector system based on simian immunodeficiency virus (SIV) that efficiently transfers genes into cytokine mobilized, peripheral blood stem cells of rhesus macaques yielding approximately 20% genetically modified cells following hematologic reconstitution.
In Specific Aim 1, experiments are proposed with a goal of achieving a high level of transduction efficiency into repopulating stem cells from steady state bone marrow while limiting vector copy number to 1 or as few as possible per transduced cell. Efforts will be also be made to highly enrich stem cells so that the transduced cells infused can be limited to those which have the potential to contribute to long-term repopulation. To evaluate the safety of stem cell-targeted gene transfer, all transplanted animals will be monitored long-term for the behavior of clones of hematopoietic cells containing a vector genome which has integrated near a proto-oncogene.
In Specific Aim 2, experiments are designed to determine whether drug selected hematopoietic stem cells will generate erythroblasts expressing higher levels of vector encoded gamma-globin than non-selected stem cells. Optimized globin vectors and vectors designed to activate the endogenous gamma genes will be evaluated in cultured erythroid cells in an effort to find a vector design which ensures equal to or >20% HbF. Promising therapeutic globin vectors will be tested in vivo in the rhesus model.
In Specific Aim 3, we will test the hypothesis that the vectors containing beta-globin locus control elements will exhibit lineage-restricted activation of surrounding genes in cell lines and primary murine hematopoietic cells that can be blocked or diminished by an insulator. Overall, our research is focused on developing effective approaches for gene therapy of sickle cell disease while simultaneously evaluating the safety of these approaches.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL053749-13
Application #
7280439
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2006-09-01
Budget End
2007-08-31
Support Year
13
Fiscal Year
2006
Total Cost
$312,636
Indirect Cost
Name
St. Jude Children's Research Hospital
Department
Type
DUNS #
067717892
City
Memphis
State
TN
Country
United States
Zip Code
38105
Zhao, Hui Fen; Abraham, Allistair; Kim, Yoon-Sang et al. (2017) Lentiviral Transfer of ?-Globin with Fusion Gene NUP98-HOXA10HD Expands Hematopoietic Stem Cells and Ameliorates Murine ?-Thalassemia. Mol Ther 25:593-605
De Ravin, Suk See; Wu, Xiaolin; Moir, Susan et al. (2016) Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency. Sci Transl Med 8:335ra57
Abraham, Allistair; Kim, Yoon-Sang; Zhao, Huifen et al. (2016) Increased Engraftment of Human Short Term Repopulating Hematopoietic Cells in NOD/SCID/IL2r?null Mice by Lentiviral Expression of NUP98-HOXA10HD. PLoS One 11:e0147059
Wielgosz, Matthew M; Kim, Yoon-Sang; Carney, Gael G et al. (2015) Generation of a lentiviral vector producer cell clone for human Wiskott-Aldrich syndrome gene therapy. Mol Ther Methods Clin Dev 2:14063
Pestina, Tamara I; Hargrove, Phillip W; Zhao, Huifen et al. (2015) Amelioration of murine sickle cell disease by nonablative conditioning and ?-globin gene-corrected bone marrow cells. Mol Ther Methods Clin Dev 2:15045
Zhou, Sheng; Bonner, Melissa A; Wang, Yong-Dong et al. (2015) Quantitative shearing linear amplification polymerase chain reaction: an improved method for quantifying lentiviral vector insertion sites in transplanted hematopoietic cell systems. Hum Gene Ther Methods 26:4-12
Urbinati, Fabrizia; Hargrove, Phillip W; Geiger, Sabine et al. (2015) Potentially therapeutic levels of anti-sickling globin gene expression following lentivirus-mediated gene transfer in sickle cell disease bone marrow CD34+ cells. Exp Hematol 43:346-351
Treanor, Louise M; Zhou, Sheng; Janke, Laura et al. (2014) Interleukin-7 receptor mutants initiate early T cell precursor leukemia in murine thymocyte progenitors with multipotent potential. J Exp Med 211:701-13
Griffith, Linda M; Cowan, Morton J; Notarangelo, Luigi D et al. (2014) Primary Immune Deficiency Treatment Consortium (PIDTC) report. J Allergy Clin Immunol 133:335-47
De Ravin, Suk See; Gray, John T; Throm, Robert E et al. (2014) False-positive HIV PCR test following ex vivo lentiviral gene transfer treatment of X-linked severe combined immunodeficiency vector. Mol Ther 22:244-245

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