The primary purpose of this research program is to develop a highly innovative approach for the treatment of sickle cell disease (SCD) by repairing the causative mutation in hematopoietic stem cells (HSCs) from affected individuals. SCD is due to a single point mutation in the beta-globin gene that gives rise to aberrant hemoglobin. Traditional therapeutic approaches (chronic blood transfusion, bone-marrow transplantation, and hydroxyurea) require persistent and intensive treatment regimes yet fail to eliminate treatment-related consequences such as end-organ damage, viral infections, progressive liver fibrosis, cardiac disease, and endocrine dysfunction. More recently, individuals afflicted with SCD have received grafts of allogeneic HSCs from normal donors. However, this approach is hampered by major problems including (i) an essential requirement for an HSC donor with an HLA type closely matching the recipient in order to minimize the incidence of graft-versus-host-disease (GVHD), and (ii) immunosuppressive drugs must be administered to allow retention of the HSC graft. At present, allogeneic bone marrow transplantation procedures have a morbidity and mortality rate of about 20%. Here we present a unique approach to treating SCD that avoids the problems described above. Using a process we term """"""""gene correction,"""""""" we propose to permanently repair the genetic lesion in the endogenous gene of HSCs taken from the affected individual and then return these autologous HSCs to the patient. This novel methodology involves selective replacement of a point mutation in the endogenous human beta-globin gene with wild type sequence using facilitated homologous recombination. Such correction involves introducing a double stranded DNA break in the mutant beta-globin gene using an enzyme that can be engineered to selectively cleave genomic DNA at virtually any desired location. The break is then repaired using the cell's own homologous recombination machinery by copy-pasting wild-type genetic information from a donor DNA molecule introduced into the cell. The corrected HSCs would then be reintroduced back into the donor. This autologous therapy will not require the use of HLA-matched donors, and thus will be available to a much larger pool of patients and avoid the need for immunosupression. While this study focuses on SCD, this approach represents a dramatic advance for the repair of any genetic point lesion.
