| Budget Amount *help |
¥3,700,000 (Direct Cost: ¥3,700,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
Fiscal Year 1999: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
Chronic granulomatous disease(CGD) is an inherited disorder of host defense against microbial infections caused by defective activity of the phagocyte NADPH oxidase. I had purified p47- and p67-phox protein and cloned the gene of this enzyme complex and analyzed the genes of patients with chronic granulomatous disease. Since 1994, I have involved in the development of retrovirus vector (Ha-MDR-IRES-gp91/PA317, Ha-MDR-IRES-p67/PA317 and Ha-MDR-IRES-p47/PA317) for gene therapy to the patients. The following problems in the clinical application have been raised in our retrovirus ; 1)low and short expression of the interested protein, 2)low virus titer of the retrovirus, 3)inactivaton of transcription etc. MFGS-gp91/293/SPA that had been developed in the cooperative work had a problem about inactivation of transcription etc. These low protein expression efficiency after gene introduction and unstable expression might be caused by the inactivation of the methylation of the virus promoter. B
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y these experience, we employed MND retorvirus vector which is tolerate for methylation of the virus promotor area developed. We also employed PAMP51 cell reported as a high titer retrovirus producer cell in stead of PA317 producer cell. MND-gp-91 plasmid was transfected in PAMP51 cell and several high titer retrovirus producer cell were cloned. In this cloning procedure, we could only use FACS analysis by 7D5 and gp91-phox monoclonal antibody because this vector does not have the selection marker. In more than 200 MND-gp-91/PAM51 clones, the highest virus titer was 1 2 X 10^5/ml which was just 2-3 times higher than MND-gp91/PA317. This titer was 50〜100 times lower than MFGS-gp91/293/SPA vector(1-2X10^7/ml). MND-gp91/PAM51 retrovirus recover superoxide generating activity only 4 5% of normal after transduction to the gp91-phox deficient patient B cell. It was not possible to confirm the tolerate effect on MND vector against inactivation by methylation.
Apart from this gene therapy study, we reported additional kindred in whom an IFN-γ-dependent increase in neutrophil superoxide production was observed in three affected patients. The defect in the CYBB gene for gp91-phox was identified as an otherwise silent mutation adjacent to the third intron of CYBB gene that alters mRNA splicing. By molecular analysis, we found significant differences in the splicing pattern of CYBB gene transcripts in patient neutrophils between 1 and 25 days after administration of INF-γ. Furthermore, a complete transcript containing the missing exons could be detected in all specimens after the treatment. The changes in the splicing pattern of the transcripts and the prolonged effect on superoxide generating ability of patient neutrophils indicate that INF-γ induced a partial correction of the abnormal splicing of CYBB gene transcripts in myeloid progenitor cells. Less
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