The B-thalassemias are the most common single gene defect in humans and result from absent or decreased B-globin synthesis, leading to severe anemia. Patients with B-thalassemia major are treated with life-long transfusions. Bone marrow transplantation can be curative, but is limited to a few with matched donors, and has potentially serious complications. Replacement of a normal B-globin gene into hematopoietic stem cells (HSCs) can potentially correct the disorder permanently, avoiding the complications associated with a transplant. With the advent of better vectors, improved gene transfer techniques and a better understanding of stem cell and vector biology, gene therapy is going from the bench to the bedside, in diseases like SCID and hemophilia B. 'Globin' gene therapy has suffered from problems of vector instability, low titers and variable expression. The recently developed lentiviral vectors transduce the non-dividing HSCs and stably export large genomic fragments by unique RNA export mechanisms, imparting stability to globin vectors. Self-inactivating (SIN) lentiviral vectors are even more advantageous: the viral LTR is deleted upon integration into cells, completely inactivating viral transcription. This feature is ideal for the expression of a highly lineage-restricted gene such as globin, and additionally improves their bio-safety. We have recently shown remarkably lineage-specific and long-term expression of GFP and gamma-globin from SIN lentiviral vectors in mouse erythroleukemia (MEL) cells, primary murine and human cells. We propose to capitalize on these findings by examining the capabilities of SIN lentiviral vectors to carry the human B-globin gene and erythroid regulatory elements for gene transfer into HSCs that results in stable, lineage-specific and sustained expression of B-globin in RBCs.
The aims of the study are to: 1) Develop SIN-lentiviral vectors carrying the human B-globin gene under control of erythroid regulatory elements, and screen them in MEL cells for stable transmission and high level expression. 2) Determine the efficacy, lineage specificity and long term expression of B-globin SIN lentiviral vectors in vivo, in thalassemic mice. 3) Determine the gene transfer capacity and efficacy of B-globin SIN lentiviral vectors in the RBC progeny of human thalassemia progenitor cells, using a unique model of human RBC production developed in our laboratory from hematopoietic progenitor cells. Together, these aims comprise a focussed research program to produce therapeutic and sustained levels of B-globin in human thalassemia RBCs, and form the basis for future preclinical studies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL070135-04
Application #
6879019
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Evans, Gregory
Project Start
2002-05-08
Project End
2007-03-31
Budget Start
2005-04-01
Budget End
2006-03-31
Support Year
4
Fiscal Year
2005
Total Cost
$299,200
Indirect Cost
Name
Children's Hospital of Los Angeles
Department
Type
DUNS #
052277936
City
Los Angeles
State
CA
Country
United States
Zip Code
90027
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Urbinati, Fabrizia; Arumugam, Paritha; Higashimoto, Tomoyasu et al. (2009) Mechanism of reduction in titers from lentivirus vectors carrying large inserts in the 3'LTR. Mol Ther 17:1527-36
Arumugam, Paritha I; Higashimoto, Tomoyasu; Urbinati, Fabrizia et al. (2009) Genotoxic potential of lineage-specific lentivirus vectors carrying the beta-globin locus control region. Mol Ther 17:1929-37
Perumbeti, Ajay; Higashimoto, Tomoyasu; Urbinati, Fabrizia et al. (2009) A novel human gamma-globin gene vector for genetic correction of sickle cell anemia in a humanized sickle mouse model: critical determinants for successful correction. Blood 114:1174-85
Wang, Daren; Zhang, Wei; Kalfa, Theodosia A et al. (2009) Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome. Proc Natl Acad Sci U S A 106:19958-63
Arumugam, Paritha I; Scholes, Jessica; Perelman, Natalya et al. (2007) Improved human beta-globin expression from self-inactivating lentiviral vectors carrying the chicken hypersensitive site-4 (cHS4) insulator element. Mol Ther 15:1863-71
Higashimoto, T; Urbinati, F; Perumbeti, A et al. (2007) The woodchuck hepatitis virus post-transcriptional regulatory element reduces readthrough transcription from retroviral vectors. Gene Ther 14:1298-304
Madigan, Catherine; Malik, Punam (2006) Pathophysiology and therapy for haemoglobinopathies. Part I: sickle cell disease. Expert Rev Mol Med 8:1-23
Urbinati, Fabrizia; Madigan, Catherine; Malik, Punam (2006) Pathophysiology and therapy for haemoglobinopathies. Part II: thalassaemias. Expert Rev Mol Med 8:1-26
Malik, Punam; Arumugam, Paritha I (2005) Gene Therapy for beta-thalassemia. Hematology Am Soc Hematol Educ Program :45-50

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