103. PGK and CMV Promoters Exert the Strongest Activity in Lentiviral Gene Transduction of Myeloid Cells Including Mature Neutrophils
Authors: Yasushi Soda, Kenzaburo Tani, Xiaojin Li, Yuansong Bai, Seok-Goo Cho, Muneyoshi Futami, Minghan Chen, Seiichiro Kobayashi, Hiroyuki Miyoshi, Hideki Sumimoto, Shoichi Ohga, Toshiro Hara, Arinobu Tojo, Shigetaka Asano
Journal: Molecular Therapy (2005)

Gene therapy for the congenital deficiencies of neutrophil function such as chronic granulomatous disease (CGD) has been considered to be a curative approach. Lentiviral vectors are considered to be one of the most appropriate vectors for CGD gene therapy. In this study, we evaluated the activity of several promoters in myeloid cells including mature neutrophils. The VSV-pseudotyped lentiviral vectors (HIV vectors) used in this study consisted of the HIV-1 construct and GFP genes driven by promoters of cytomegalovirus (CMV), phosphoglycerokinase (PGK), elongation-factor 1|[alpha]| subunit (EF-1|[alpha]|), CAG compound (CAG) or human neutrophil specific |[alpha]|-defensin (DEF). Since the difference of promoter influences the transgene expression level and consequently affect the GFP titer, we adopted the DNA titer, which was not influenced by the internal promoter activity. At first, we transduced those HIV vectors into acute promyelocyte leukemia NB4 cells. The EF-1|[alpha]| and PGK were the strongest promoters in those cells, while the activities of these promoters became weak after the differentiation induced by all-trans retinoic acid. In contrast, the CMV promoter activity was the strongest in the differentiated cells. CAG and DEF showed similar activity patterns as CMV and EF-1|[alpha]|, respectively, but the expression level was low. Next, we transduced those HIV vectors into the CD34+ cord blood cells (CBCs) at an MOI of 1000, and these cells were cultured in the presence of cytokine cocktail (SCF, TPO, IL-3 and G-CSF) for 7 days followed by the 8 days culture with G-CSF. 7 days after transduction, the majority of cells ranged from promyelocytes to metamyelocytes, and the GFP+ rates of these cells transduced with the HIV vectors with PGK, EF-1|[alpha]|, CMV and DEF were 70%, 60%, 60% and 40%, respectively. Those GFP expressions were observed more strongly in CD34- cells than in CD34+ cells. As for the HIV vector with CAG, transduction was performed at an MOI of 100, because we could not produce high titer vectors, and the transduction efficiency was 50%. 15 days after transduction, 50 to 60% of cells were mature neutrophils and their GFP expression levels became weak regardless of vectors. However, the GFP+ rate with PGK and CMV promoter were relatively maintained with 50 and 40%, respectively. In addition, fluorescence intensity of GFP+ transduced with the HIV vector with CMV was the highest. Furthermore, the transduction of any kind of the HIV vectors did not affect significantly the growth, viability and functions of the differentiated neutrophils. In conclusion, as far as we could survey, PGK and CMV promoters were considered to be the most suitable for the lentiviral gene transduction to obtain appropriate gene expression of therapeutic level in both of immature and mature myeloid cells. We are now investigating the usefulness of those vectors for functional correction of neutrophils from CGD patients in vitro.