178. Site-Specific Base Alteration in the Promoter Region of the Human Gamma Globin Gene by Single-Stranded Oligonucleotides for Gene Therapy of Sickle Cell Disease
Authors: W. Yin, B. T. Kren, C. J. Steer
Journal: Molecular Therapy (2004)

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The human –globin chain has shown greater capacities for inhibiting the polymerization of sickle (S) hemoglobin (Hb) than the –globin chain itself. Moreover, increasing the synthesis of fetal (F) Hb in adults by specific pharmacologic agents has been developed as an alternative strategy to ameliorate sickle cell anemia and –thalassemia. Unfortunately, their clinical application is hampered by their considerable toxicity. Some naturally occurring point mutations in the promoters of A– and G– globin genes are associated with increased HbF in adult erythroid cells. In particular, the C to G mutation at –202 in the G– gene promoter increases the level of HbF up to 15–25% of total hemoglobin, which is sufficient for ameliorating the clinical manifestations of sickle cell anemia. Thus, reactivation of –globin gene expression by targeted mutagenesis of specific sites provides a novel strategy for gene therapy of sickle cell disease. Short (45–100 nt) single-stranded oligonucleotides (SSOs) have been shown to mediate site-specific alteration of single base pairs in both episomal and chromosomal target genes in mammalian cells. The exchange reaction can result in either repair of a lesion or creation of a mutation and is mediated through endogenous DNA repair pathway(s). The aim of this study was to determine if SSO directed DNA repair could introduce specific single base changes in the G– gene promoter region. Forty-five mer SSOs were designed homologous to the G– globin promoter target site –202 except for the middle base which was designed to induce a C to G mutation. This single base alteration at nt –202 also changes the wild-type (wt) Ban II restriction endonuclease site to an Ehe I site, providing a restriction fragment length polymorphism (RFLP) for detection of the desired C to G change. To increase resistance to intracellular nucleases, the SSOs were synthesized with either amine or phosphorothioate linkages at both ends. At various times following transfection of human cell lines with the modified SSOs, genomic DNA was isolated from transfected and control cells and subjected to Ban II digestion. After agarose gel separation of the digested DNA, the genomic DNA from 2.3–3 kb was excised, purified and used as the template for PCR amplification of the G– promoter region spanning the target site. Only cells treated with the SSOs exhibited the desired site-specific C to G change detected by PCR-RFLP. DNA sequencing confirmed that this was the only nt change introduced in the region targeted by the SSO. Moreover, we developed a sensitive and reliable detection system which is suitable for detection of >0.1% point mutations in a wt background. Finally, our methodology for detection indicates that the C to G change was not a PCR artifact. In particular, electrophoretic separation of the digested genomic DNA was done prior to the PCR amplification for the RFLP analysis. In conclusion, 45 mer end-modified SSOs can be used to generate site-specific point mutations in the G– globin gene promoter region to specifically increase HbF expression in adult erythroid cells. This novel strategy provides a gene therapy approach for sickle cell disease and other hemoglobinopathies, independent of correcting the primary genetic lesion in the Hb gene.