Lentiviral vectors for gene therapy for beta-thalassemia

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DESCRIPTION (provided by applicant): 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.