Correction of globin gene mutations by gene targeting

Washington University, Saint Louis MO

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Abstract

DESCRIPTION (provided by applicant): Beta-thalassaemia and sickle cell anemia are severe illnesses that result in considerable morbidity and early mortality for most patients. Conventional therapy prolongs life, but does not cure these disorders. Gene therapy has not yet been effective, due to large variations in the outputs of beta -globin genes when they are randomly integrated into host cell genomes. Furthermore, this approach causes random mutations in the genome, which could potentially lead to the disregulation of oncogenes. For these reasons, the specific correction of mutant beta -globin genes may represent the optimal means for gene therapy. Subtle mutations can be corrected with homologous recombination, where wild type DNA sequences can replace mutant genes in a specific locus. This process has not been used in gene therapy trials to-date, because it is extremely inefficient. Recent experiments in our lab suggest that embryonic stem cells might perform homologous recombination more efficiently if they are given extra amounts of the proteins important for the homologous recombination process. In this proposal, we will determine whether any of the homologous recombination proteins are rate-limiting for this process in embryonic stem cells, and we will extend that information to primary bone marrow cells. We hope to ultimately determine whether this approach could be used to perform homologous recombination-mediated gene "correction" of mutant human beta -globin genes.