Human hematopoietic stem cell gene therapy

Many heritable or nonheritable diseases (e.g. sickle cell disease, severe combined immunodeficiency, bone marrow suppression due to chemotherapy) that affect the function of the blood system can potentially be treated with genetically modified blood stem cells. Glass needle-mediated microinjection of primary human blood stem cells is a novel approach that allows efficient delivery of a transgene directly into the nuclei of cells without causing the loss of stem cell activity. In this study, microinjection was examined for its efficacy in treating two different types of blood system diseases: bone marrow suppression due to chemotherapy and sickle cell disease. We first evaluated the efficacy of protecting human blood stem cells from a variety of chemotherapeutic drugs by modifying cells with an episomal construct expressing both the human MDR-1 and the human MGMT genes. Results indicated that the episomal construct co-expressing MDR-1 and MGMT protected the transfected K562 cells from combination chemotherapy (Taxol + BCNU). We were able to deliver the episomal vector into primary CD34+ cells via microinjection. However, the episomal construct was rapidly lost from the injected cells, which prevented us from further analyzing the transfected CD34+ cells. We then evaluated the efficacy of microinjection-mediated targeted gene conversion in human blood stem cells via chimeric RNA/DNA oligonucleotides designed to direct an A to T substitution within codon 6 in the human β-globin gene. This A to T nucleotide exchange introduces the single site mutation responsible for sickle cell anemia. Gene conversion was detected in the progeny of both CD34+ and Lin−CD38− cells that were cultured for at least 4 weeks. In addition, gene conversion was detected in the erythroid progeny of Lin−CD38 − cells at both the DNA and mRNA level. Conversion rates as high as 10–15% in some samples were confirmed by allele-specific PCR and sequence analysis of genomic DNA from the progeny of microinjected Lin−CD38− cells. Given that as few as 10% normal hematopoietic cells are sufficient to keep patients free of sickle cell disease, the level of conversion we have achieved may well be of therapeutic benefit in patients with sickle cell disease.