LNCX Retroviral Vector를 이용한 CD34+골수세포 neoR 유전자 Transfer

Abstract

Background: The hematopoietic stem cells have been one of the major targets for designing human gene therapy of genetic disorders and malignant diseases. As a step toward the clinical application of gene transfer into hematopoietic stem cells, we have infecteded LNCX retroviral vector into purified CD34+ bone marrow cells and examined the effect of fibronectin on transduction. Methods : CD34+ bone marrow cells were purified in 3 normal marrow donors by immunomagnetic microbead methods, and subsequently primed with stem cell factor(SCF), interleukin-3(IL-3), interleukin-6(IL-6) for 48 hours. After priming, CD34+ bone marrow cells were cultured at a density of 2× 10 5 5mL in supemant containing neoR gene inserted LNCX retroviral vector for 72 hours. Cultures were supplemented with protamine, SCF, IL-3 and IL-6. Every 24 hours, cells were spun down and resuspended in fresh retroviral supernant, protamine and growth factors. After retroviral-mediated gene transfer, clonogenic assay and polymerase chain reaction(PCR) analysis were taken. Results : 1) At clonogenic assay, hematopoietic colonies were found in all LNCX retroviral-infected CD34+ cells-plated culture, irrespective of use of G4l8(0.9mg/mL), but in Mock-infected CD34+ cells plated culture, any colonies were not observed with use of G4l8. After retroviral-mediated gene transfer, no major shift in colony composition was found. 2) PCR analysis revealed amplified neoR gene band in LNCX retroviral-infected colonies, but not in Mock-infected colonies. 3) LNCX retroviral-transduction efficiency(neoR %) was 14.8% in G418 clonogenic assay. Transduction efficiency was increased into 20.8% by use of fibronectin. Conclusion : We showed effective LNCX retroviral-infection into normal CD34+ bone marrow cells, which were separated with immunomagnetic microbead methods, and primed with SCF, IL-3 and IL-6 in fibronectin-coated culture flask. These preclinical data have contributed to the basis of ongoing gene transfer into hematopoietic stem cells.