This renewal proposal describes experiments for which 5 years continuing support is requested. The project, about to enter its 10th year of funding, continues to be centered upon the goal of establishing methods for the precise correction of beta-globin defects by gene targeting (homologous recombination). During the preceding period of support, the Applicant and his colleagues achieved a number of technological advances, and made a number of interesting observations that emphasize the delicacy and complexity of the system. They devised a relatively efficient and precise method for inserting a desired gene sequence, at a reasonable frequency, within the beta-globin gene. This """"""""plug and socket"""""""" methodology appears to be more efficient and reliable than most other methods. They have also observed, however, selectable marker genes, needed to enrich the population specifically for cells undergoing the desired recombination events, also carry the potential to derange expression of the target gene locus, possibly by competition of the selectable promoter for critical enhancer sequences, such as the LCR. Nearby genes were also adversely affected, suggesting that this methodology could have deleterious and unintended consequences if used therapeutically. The present proposal has three Specific Aims. Each is intended to address strategic issues raised by results obtained during the current period of funding. The first Specific Aim will focus upon further refinement of the plug and socket system in an effort to achieve controllable and """"""""harmless"""""""" targeted recombination, especially as it pertains to the effects of targeting upon function upon the endogenous beta-globin promoter. The Investigator proposes to utilize a number of constructs within which different promoters and different configurations of promoters are combined in an effort to alter the negative impact of targeting upon expression of the endogenous beta-globin gene. The second Specific Aim will employ the HPRT locus on the X chromosome, which can be manipulated readily as a positive or negative selectable marker, as a target site. This gene will be used for development of methods to give cells carrying the homologously recombined gene sequences a positive growth advantage after bone marrow transplantation. The Investigator proposes to start with introduction of the genes for BCL-2, an inhibitor of apoptosis, and the truncated erythropoietin receptor, as sequences to be introduced into the targeted gene. These should allow cells carrying the recombined locus to enjoy a proliferative advantage during hematopoiesis. Preliminary experiments suggest that animals carrying these cells do not exhibit a propensity for neoplastic diseases. After an appropriate combination of """"""""advantage genes"""""""" for targeting has been identified, the investigator will attempt to utilize these for targeting of the beta-globin locus. The third Specific Aim, clearly the most speculative, will attempt to move the targeting technology from MEL cells and embryonic stem cells (ES), in which the methodologies have been perfected, to hematopoietic stem cells. The ultimate goal of these studies is to achieve clinically usable efficiencies of targeting into pluripotent hematopoietic stem cells. In the preliminary stages of these experiments, the Investigator proposes to utilize unfractionated stem cells, and to accept transfer into more committed progenitors. When methods for transfer and update of the genes have been optimized, he will attempt to obtain more highly purified populations.
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