Small fragment homologous replacement-mediated modification of genomic β-globin sequences in human hematopoietic stem/progenitor cells

An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A -> T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained :51 fragment per cell. Control studies mixing genomic DNA from nontransfected cells with varying amounts of the targeting SDFs did not indicate any PCR amplification artifacts due to the presence of residual SDF during amplification. RNA was analyzed after DNase treatment, thus eliminating the potential for SDF contamination. Stable SFHR-mediated conversion of normal (beta(A)) to sickle (beta(S)) globin was detected at frequencies up to 13% in cells harvested 30-45 days posttransfection. The minimum conversion efficiency ranged from 0.2 to 3%, assuming modification of at least one cell per experiment showing conversion. Conversion of sickle (beta(S)) to normal (beta(A)) globin was detected up to 10 days posttransfection in lymphoblastoid cells from a sickle cell patient. These studies suggest that SFHR may be effective for ex vivo gene therapy of sickle cells in a patient's HSPCs before autologous transplantation.