Studies for Gene Therapy of Sphingolipidosis

Gene analysis of Japanese lipidodes patients：We analyzed gene from various Japanese patients with lipidoses and found that the distribution of gene mutations was quite different from that from other ethnic groups.Gene therapy：The deficiency of humanβ-glucuronidase(HBG)results in MPS type VII(Sly Syndrome)。In this study，we tested the ability to target CD18/CD11b positive cells with gene therapy for murine MPS VII.We harvested bone marrow cells from syngeneic normal mice and cultivated in CSF-1 containing medium for 2 weeks.More than 95%of the cells were double positive for CD18/CD11b by flowcytometry.We gave2×10 I D16 I D 1 cells to the non-myeloablated Sly mouse.One week post-transplantation，donor cells populated liver and spleen。HBG activity increased from 0.9±0.7 to 28.4±12.5 u/mg and 0.7±0.4 to 29.7±23.1 u/mg in liver and spleen respectively.However，the pathology was unchanged.HBG activity in liver and spleen decreased b5weeks to 3.7±1.5 and 2.3±0.5 respectivel…More y，but the pathological improvements were significant and glycosaminoglycan storage was largely cleared.Next，the CD18/CD11b cells were collected from Sly mouse by the method above，transduced by HBG expressing retrovirus(MFG-HBG)，and given to non-myeloablated Sly mouse。By5weeks post-transplantation，HBG activity was only marginally above the control level.However，many HBG positive cells were observed and pathological improvement was significant.These data suggest that CD18/CD11b cells transplantation is promising for treatment of murine MPS VII without myeloablation，and CD18/CD11b cells may be a good targets for gene therapy for Sly syndrome.Gene transfer to neuronal cells：Because significant prenatal pathology occurs in genetic diseases，postnatal therapy is often not sufficient to correct them，especially if damage hocas curred in the central cervous。In such diseases，prenatal treatment will be required.Adenovirus mediated gene transfer may be useful for this purpose because of its wide host range and efficient gene transfer to various organs。However，most of the gene transfer studies have been carried out at a relatively late stage of embryogenesis。In this study，a recombinant adenovirus expressing the E.coli lacZ gene(AxCALacZ)was administered into the rat embryos at the 8th to 12th day of gestation。The results of the study revealed that the lacZ gene was expressed in many different organs including liver，heart，skin and brain.These results suggest that the adenovirus vector may be useful for the prenatal gene therapy of many genetic diseases.Less：Less

Gene analysis of Japanese lipidodes patients :We analyzed gene from various Japanese patients with lipidoses and found that the distribution of gene mutations was quite different from that from other ethnic groups.Gene therapy :The deficiency of human β-glucuronidase(HBG) results in MPS type VII(Sly syndrome). In this study, we tested the ability to target CD18/CD11b positive cells with gene therapy for murine MPS VII. We harvested bone marrow cells from syngeneic normal mice and cultivated in CSF-1 containing medium for 2 weeks. More than 95% of the cells were double positive for CD18/CD11b by flowcytometry. We gave 2×10ィイD16ィエD1 cells to the non-myeloablated Sly mouse. One week post-transplantation, donor cells populated liver and spleen. The HBG activity increased from 0.9±0.7 to 28.4±12.5u/mg and 0.7±0.4 to 29.7±23.1u/mg in liver and spleen respectively. However, the pathology was unchanged. The HBG activity in liver and spleen decreased b 5 weeks to 3.7±1.5 and 2.3±0.5 respectivel … More y, but the pathological improvements were significant and glycosaminoglycan storage was largely cleared. Next, the CD18/CD11b cells were collected from Sly mouse by the method above, transduced by HBG expressing retrovirus(MFG-HBG), and given to non-myeloablated Sly mouse. By 5 weeks post-transplantation, HBG activity was only marginally above the control level. However, many HBG positive cells were observed and pathological improvement was significant. These data suggest that CD18/CD11b cells transplantation is promising for treatment of murine MPS VII without myeloablation, and CD18/CD11b cells may be a good targets for gene therapy for Sly syndrome.Gene transfer to neuronal cells :Because significant prenatal pathology occurs in genetic diseases, postnatal therapy is often not sufficient to correct them, especially if damage has occurred in the central nervous system. In such diseases, prenatal treatment will be required. Adenovirus mediated gene transfer may be useful for this purpose because of its wide host range and efficient gene transfer to various organs. However, most of the gene transfer studies have been carried out at a relatively late stage of embryogenesis. In this study, a recombinant adenovirus expressing the E. coli lacZ gene (AxCALacZ) was administered into the rat embryos at the 8th to 12th day of gestation. The results of the study revealed that the lacZ gene was expressed in many different organs including liver, heart, skin and brain. These results suggest that the adenovirus vector may be useful for the prenatal gene therapy of many genetic diseases. Less