Verbatim): Sickle cell anemia is one of the commonest inherited diseases in humans, characterized by a severe chronic hemolytic anemia with an unpredictable course. While current forms of chemotherapy do not represent a radical treatment, the use of bone marrow replacement is limited by complications of allogeneic transplantation and the need for aggressive conditioning regimens. Thus, the goal of this proposal is to develop a treatment for severe hemoglobinopathies that integrates a genetic correction in autologous hematopoietic stem cells (HSC) with a reasonable transplantation strategy. The approach we propose is based on efficient lentiviral-mediated transfer of a wild-type globin gene in cord blood or peripheral blood stem cells, together with a selection for genetically modified cells that is applied in vivo after transplantation. In vivo selection is useful for two purposes: (1) to increase the relative representation of genetically corrected blood cells and (2) to decrease the toxicity associated with the transplantation conditioning regimen. Our recent results establish that efficient gene transfer of a modified beta-globin gene and large elements of the beta-globin LCR can be achieved using recombinant lentiviruses. We have demonstrated that (1) a large LCR greatly increases mean globin expression compared to the core elements of the LCR that were previously investigated and (2) incorporation of an insulator element into a retroviral vector increases the probability of expression at random integration sites and decreases vector silencing. The major goals of this project are: (a) to improve erythroid-specific gene expression from a virally encoded beta-globin transcription unit; (b) to compare the betaAand gammaAglobin genes in terms of their level of expression in bone marrow chimeras and their therapeutic activity in mouse models of sickle cell disease; (c) to confer a competitive advantage to the transduced HSC for repopulation of the host marrow using resistance to methotrexate as a model. We propose a detailed analysis of the function of the LCR and of the chicken globin insulator in stringent in vitro and in vivo assays that are relevant to the critical evaluation of their therapeutic potential. These studies are based on investigations in murine models of sickle cell disease and in primary human CD34+ cells of normal subjects and patients. To analyze globin gene expression and the effectiveness of drug resistance in selecting out corrected cells that express therapeutic levels of the globin transgene, we will capitalize on our ability to efficiently derive erythroid progeny from long-term cultured CD34+ cells and our mouse/human xenochimeras based on NOD-scid/scidmice. We ultimately aim to establish by direct experimental evidence that expression of the lentivirus-encoded human globin gene is sustained over time in murine and human cells in vivo and that expression of the mutant dihydrofolate reductase permits efficient in vivo selection with methotrexate/trimetrexate.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL057612-07
Application #
6755974
Study Section
Hematology Subcommittee 2 (HEM)
Program Officer
Evans, Gregory
Project Start
1996-09-30
Project End
2007-05-31
Budget Start
2004-06-01
Budget End
2007-05-31
Support Year
7
Fiscal Year
2004
Total Cost
$366,750
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Papapetrou, Eirini P; Korkola, James E; Sadelain, Michel (2010) A genetic strategy for single and combinatorial analysis of miRNA function in mammalian hematopoietic stem cells. Stem Cells 28:287-96
Papapetrou, Eirini P; Kovalovsky, Damian; Beloeil, Laurent et al. (2009) Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T cells in murine hematopoietic chimeras. J Clin Invest 119:157-68
Chang, Alex H; Stephan, Matthias T; Lisowski, Leszek et al. (2008) Erythroid-specific human factor IX delivery from in vivo selected hematopoietic stem cells following nonmyeloablative conditioning in hemophilia B mice. Mol Ther 16:1745-52
Lisowski, Leszek; Sadelain, Michel (2007) Locus control region elements HS1 and HS4 enhance the therapeutic efficacy of globin gene transfer in beta-thalassemic mice. Blood 110:4175-8
Budak-Alpdogan, Tulin; Przybylowski, Mark; Gonen, Mithat et al. (2006) Functional assessment of the engraftment potential of gammaretrovirus-modified CD34+ cells, using a short serum-free transduction protocol. Hum Gene Ther 17:780-94
Chang, Alex H; Stephan, Matthias T; Sadelain, Michel (2006) Stem cell-derived erythroid cells mediate long-term systemic protein delivery. Nat Biotechnol 24:1017-21
Samakoglu, Selda; Lisowski, Leszek; Budak-Alpdogan, Tulin et al. (2006) A genetic strategy to treat sickle cell anemia by coregulating globin transgene expression and RNA interference. Nat Biotechnol 24:89-94
Tabar, Viviane; Panagiotakos, Georgia; Greenberg, Edward D et al. (2005) Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain. Nat Biotechnol 23:601-6
Sadelain, Michel; Lisowski, Leszek; Samakoglu, Selda et al. (2005) Progress toward the genetic treatment of the beta-thalassemias. Ann N Y Acad Sci 1054:78-91
Sadelain, Michel; Rivella, Stefano; Lisowski, Leszek et al. (2004) Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease. Best Pract Res Clin Haematol 17:517-34

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