Cooleys anemia (b-Thalassemia major) and Sickle Cell Disease are caused by mutations in the b-globin genes. Severe cases can be cured by Bone Marrow Transplantation, but with a significant risk of death due to graft rejection. Gene Therapy, in which a viral vector containing a b-globin gene is inserted into patient bone marrow cells, carries none of the risks of graft rejection and could be an alternative cure for Cooleys anemia or SCD. However, current vectors contain the powerful enhancer elements of the b-globin locus control region (LCR), which raises concerns that the integration of the virus near oncogenes could lead to leukemia. ? Our long-term goal is to design gene transfer vectors for the treatment of Cooleys anemia (b-Thalassemia major) andor Sickle Cell Disease that address the three challenges confronting gene therapy for this disease: therapeutic levels of expression that do not depend on enhancer elements in the viral vector or the transcription unit, resistance to position dependent gene silencing and transduction of 20% or more of human HSC. We hypothesize that self-inactivating (SIN) vector backbones containing enhancer independent globin transcription units that are pseudotyped with an envelope that recognizes abundant receptors on HSC would be the optimal system for gene therapy of these diseases. We have developed a novel strategy in which the enhancer independent Slc4a1 or erythroid ankyrin promoters are used to express the g-globin gene in a SIN lentivirus vector. Upon integration this provirus has no viral promoter and no enhancer elements. To protect against gene silencing, the Slc4a1g-globin transgene is flanked in the vector by distinct combinations of the chicken b-globin insulator, andor barrier elements from the human ankyrin and a-spectrin gene loci discovered in our lab. The ankyrin promoter carried with it its own barrier element. In preliminary experiments in mice, the Slc4a1hg-globin vectors expressed g-globin at levels of as high as 17% of the level of endogenous mouse a-globin expression, while the ankyrin vectors express at about 8%. For our patient studies we will pseudotype our lentivirus vector with the Feline Leukemia Virus type C (FeLV-C) envelope that recognize an abundant receptor expressed on human HSC.? The objective of this proposal is to test these novel vectors in mouse models and in cells from Cooleys anemia patients to determine whether this strategy safely and efficiently deliver and express therapeutic levels of g-globin in erythroid cells. We plan to pursue the following two specific aims:? 1. Evaluate the efficiency of transduction and gene expression of lentiviral vectors containing either the ankyring-globin or a Slc4a1-driven human g-globin gene flanked with distinct barrier elements. ? The working hypothesis for this aim is that these lentiviruses will safely allow for high HSC transduction efficiency and drive therapeutic levels of hg-globin without the need for enhancers.? 2. To test the best vector in primitive human stem and progenitor cells from Cooleys anemia or SCD patients using the fetal sheep transplantation model.? We believe that these studies will translate directly into safe and effective gene therapy for the two most common inherited hemoglobinopathies. ? ? Specific Aim 1: Evaluate the efficiency of transduction and gene expression of lentiviral vectors containing a Slc4a1-driven human -globin gene flanked with distinct barrier elements.? We have identified sequence modifications in the ankyrin promoter activity that increase expression in transient transfection and in vitro transcription assays that could increase the output of ANK-globn vectors beyond the 8% of endogenous mouse -gamma level we reported previously. These new promoters will be added to our prototype vectors and evaluated in the mouse models of beta thalassemia and SCD. The levels of human g-globin mRNA and protein will be determined by RNase protection and HPLC respectively. For the Slc4a1 vectors our basic lentiviral design consists of a 1.7 kb mouse Slc4a1 promoter linked to the human A-globin (Slc4a1h-globin) gene flanked by distinct barrier elements. We have previously shown that the chicken -globin 5 hypersensitive site 4 (ch5HS4) insulator protects the Slc4a1h-globin transgene from position effect variegation in transgenic mice. We and others have found that vectors containing two internal copies of ch5HS4 were prone to recombination and decreased virus titer, and similarly, inserting ch5HS4 into the 3 LTR also decreased titer significantly. The barrier-flanked Slc4a1-globin has been inserted into a HIV-1-based SIN lenitvirus, in which the 3LTR promoter and enhancer elements are deleted to preclude LTR-driven oncogene transcription. ? Specific Aim 2: To test the best vector in primitive human stem and progenitor cells from Cooleys anemia or SCD patients using the fetal sheep transplantation model. To investigate the therapeutic potential of the Slc4a1-globin vectors in human -thalassemia, we will transduce human CD34 stem and progenitor cells obtained from bone marrow of -thalassemia patients. We have previously shown that oncoretroviruses pseudotyped with the FeLV-C envelope transduce human sheep repopulating cells at much higher frequencies than the GaLV or VSV-G envelopes. We have adapted this envelope to package lentivirus vectors by deleting the R peptide in the FeLV-C envelope, similar to the successful strategies employed to adapt the MLV amphotropic envelope for packaging lentiviruses. Patient CD34 cells will be transduced and injected into preimmune fetal sheep between 55 and 60 days of gestation. The transplanted lambs will be brought to term, and two weeks after birth blood samples will be analyzed for the presence of human HSC-derived cells using an anti-human CD45 antibody. DNA extracted from human lymphoid and myeloid cells will be analyzed for integration of the provirus and integration sites. Human erythrocytes (CD45, Glycophorin A) will be isolated from peripheral blood by fluorescence activated cell sorting and the proportions of -like globin chains to -globin globin chains will analyzed by HPLC. As the lambs age, bone marrow samples will be obtained for the generation of myeloid and erythroid colony forming cells.

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National Human Genome Research Institute (NHGRI)
Intramural Research (Z01)
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