NHLBI MINORITY INSTITUTION RESEARCH SCIENTIST DEVELOPMEN

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DESCRIPTION (Adapted from applicant's abstract) Sickle cell disease is a serious hemoglobin disorder characterized by a chronic hemolytic anemia with recurrent vasoocclusive episodes, secondary complications, impaired quality of life and a reduced life expectancy. Sickle cell disease is caused by the substitution of glutamic acid with valine at the position of amino acid six in the Beta- globin chain. As a result the red blood cells synthesize sickle hemoglobin, which polymerizes under deoxygenating conditions, changing the shape of the red blood cells into sickle cells. There is no cure for sickle cell disease with the exception of allogeneic stem cell transplantation. However, this procedure has its limitations because of the difficulty in finding a suitable HLA-identical sibling donor, the high cost of bone marrow transplantation, and the high rate of morbidity and mortality. Relatively new chemotherapies like hydroxyurea has decreased the frequency of the painful episodes, and alleviated some of the disease symptoms, but does not cure the disease. There is a basic need to develop innovative therapeutic approaches to cure and treat sickle cell disease and reduce the risks of its debilitating complications. Gene therapy seems to be a good candidate to provide a cure for sickle cell disease. However, it is a formidable task due to the unavailability of an ideal vector for globin gene delivery, difficulty in transducing hematopoietic stem cells, the need for erythroid specific, high level, balanced expression of the globin genes. Retroviral and adeno-associated gene transfer based methods are being used, but they have their drawbacks like DNA rearrangements upon integration, low expression of transferred genes, and safety concerns inherent to viral vectors for human use. This proposal attempts to explore the development of alternative vectors that yield high expression of gamma- globin genes in erythroleukemia cell lines, with the potential application to replace defective globin genes in hematopoietic stem cells. Based on our studies with an autonomously replicating sequence (ARSH1), a human putative replicator, the applicant will explore the possibility of developing ARSH1- derived DNA molecules as alternative vehicles for gent transfer. These vectors will need to replicate autonomously, be stable, heritable and have long term expression of the transferred gene. The specific aims of this proposal are: 1) Construction and primary characterization of novel vectors for gamma-globin gene delivery into K-562 cells, 2) Characterization of the episomal replication activity and integrity of the transfected vector in K-562 cells, 3) Expression of the transfected vector in K-562 cells. Once this vector is developed and characterized, it can be studied in mouse hematopoietic stem cells, and later on, if these cells sustain replication and expression of the vector, the transfected stem cells could be transplanted into a sickle cell transgenic mouse model, and in vivo expression of fetal hemoglobin studied in the animal model. If successful, these studies could produce an alternative vehicle for gene therapy of sickle cell disease.