| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1996: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Research Abstract |
To test the feasibility of gene therapy for chronic granulomatous disease (CGD), we have constructed the retrovirus vector designated MFG-S-gp91 which contains gp91 phox cDNA into the cloning site of MFG.A producing cell line, psiCRIP/MFG-S gp91-12 (hereafter, gp91-12) was established by co-transfection of MFG-S-gp91 and pSV2-neo into packaging cell line psiCRIP and by subsequent limiting dilution. EBV-transformed B cell lines (LCL) were established from several X-linked CGD patients and healthy controls for the target cells of gp91phox gene transduction. LCLs were co-cultured with retroviral supernatant either from gp91-12 or a control vector producing clone for 24 hours under 32ﾟC,5% CO_2, and this step was repeated once. After 5 to 7 days from the gene transduction, superoxide production was measured using a luminol-enhanced chemiluminescence assay. In one LCL clone, this procedure could increase the superoxide production up to 20% of normal level without any selection of transduced cells, suggesting that gene transduction using this retrovirus vector system is useful for reconstitution of impaired superoxide generation in CGD cells.
To improve the efficiency of gene transduction into hemopoietic stem cells, we compared the effect of 5 different recombinant fibronectin fragments (FNFs) on gene transduction using retrovirus vector LASN and umbilical cord blood CD34 positive cells as target cells. Transfection was carried out on FNF s-precoated plates with cytokines (IL-3, IL-6 and SCF) using LASN supernatant under 37ﾟC,5%CO_2 condition. After 4 cycles of 12 hours'co-cultivation, gene transduction efficiency was assessed by colony formation assay and semi-quantitative PCR method. Of 5 FNFs tested, only CH296 which contains three major domains of FN could markedly improve the gene transduction efficiency.
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