Targeted Correction of the Human p-globin Gene. The ability to genetically manipulate human stem cells has the potential to enhance the utility of stem cell therapy for human disease. One emerging approach to targeted genome modification is the use of triplex- forming oligonucleotides (TFOs). These molecules bind to duplex DNA in a sequence-specific manner, and this binding can be used to stimulate recombination and gene correction in mammalian cells. Initial work by our group and others has demonstrated that TFOs can stimulate recombination either by delivering a tethered psoralen adduct or by the ability of triple helices, themselves, to provoke DNA repair and thereby sensitize the target site to recombination. Using a series of chemical modifications, the intracellular effectiveness of TFOs has been progressively enhanced. In this revised A2 application, we propose to develop specific TFOs and TFO analogs such as peptide nucleic acids (PNAs) for intracellular binding to the human (3-globin gene with the intention of ultimately generating an optimized reagent set for molecular correction of mutations associated with thalassemia and sickle cell anemia. We will test promising TFOs or PNAs and donor DNA molecules for the ability to correct (3-globin mutations using specially designed reporter systems in cell lines in culture. New data now demonstrate our ability to target the (3-globin gene in human cells with modified TFOs. Gene correction in mouse hematopoietic stem cells (HSCs) will be studied in cells derived from transgenic mice engineered to incorporate human p-globin gene sequences in the context of a green fluorescent protein (GFP) reporter gene. We will also test targeting of the p-globin gene in primary human CD34+ cells enriched for HSCs in assays for both TFO-targeted psoralen crosslinks and for the generation of a targeted sequence change within the p-globin gene. In this revised application, continued optimization of transfection techniques in hematopoietic stem cells and new strategies to boost gene correction frequencies will enhance these efforts. The overall hypothesis driving this application is that high-affinity, site-specific DNA binding by TFOs can be used as a tool to stimulate gene targeting in stem cells. Our long-range goal is to develop such molecules as reagents for targeted genome modification of disease-related genes in human cells. ? ? ? ?
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