Abstract
An ultimate goal of gene therapy is development of efficient methods to correct mutant genomic sequences in pathologic cells. In this study SFHR; an oligonucleotide-based gene targeting strategy; was used to convert A|[srarr]| T in codon 6 of the |[beta]|-globin gene of CD34+ Lin- Hematopoietic Stem/Progenitor Cells (HSPCs) isolated from full term umbilical cord blood. HSPCs were transfected with a defined number of a 559-bp small DNA fragment (SDF) using the AMAXA Nucleofection/Electroporation system. After growing the transfected cells in stem cell media containing EPO for different time intervals up to 32 days, RNA was extracted and DNase I-treated before further analysis. RFLP analysis of a 430-bp PCR product generated from mRNA-derived cDNA with the DdeI enzyme indicated |[beta]|A- to |[beta]|S-globin conversion. Sequencing of the 430-bp amplicon also showed the conversion A|[srarr]| T. These data are consistent with previous studies showing both conversion of |[beta]|S- to |[beta]|A-globin in SC1 cells and |[beta]|A- to |[beta]|S-globin in HSPCs after electroporation and microinjection of SDF, respectively. These studies represent a critical next step in developing SFHR as a therapy for sickle cell disease, in that , they demonstrate long-term SFHR-mediated modification of |[beta]|-globin following mass transfection by electroporation of HSPCs. This work is supported by an NIH Fellowship Training Grant DK007636 and the CPMC Research Foundation.
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