title: Retroviral-mediated transfer of the human acid sphingomyelinase cDNA: correction of the metabolic defect in cultured Niemann-Pick disease cells.
abstract: Types A and B Niemann-Pick disease (NPD) result from inherited deficiencies of the lysosomal hydrolase, acid sphingomyelinase (ASM; sphingomyelin cholinephosphohydrolase, EC 3.1.4.12). To evaluate the feasibility of somatic gene therapy for the treatment of these disorders, retroviral-mediated gene transfer was used to introduce the full-length ASM cDNA into cultured fibroblasts from two unrelated type A NPD patients. The ASM activities in these cells were less than 4% of mean normal levels, and, consequently, they accumulated approximately 3-fold elevated levels of sphingomyelin. After retroviral-mediated transfer of the ASM cDNA, ASM activities in the NPD cells increased to levels up to 16-fold those found in normal fibroblasts. In addition, the sphingomyelin content was reduced to normal levels, indicating that the vector-encoded enzyme was properly targeted to lysosomes, where it was enzymatically active and able to degrade the accumulated substrate. In situ cell-loading studies also were undertaken to evaluate the effects of retroviral-mediated gene transfer on the pathology of NPD fibroblasts. When a pyrene derivative of sphingomyelin was introduced into the lysosomes of cultured fibroblasts from a type A NPD patient by using apolipoprotein E-mediated endocytosis, only approximately 6% of the delivered substrate was degraded. In contrast, normal cells and NPD cells transduced (i.e., "corrected") by retroviral-mediated gene transfer could degrade approximately 80% of the delivered sphingomyelin. These results provided further evidence that retroviral-mediated gene transfer may be used to correct the pathology of NPD cells. Cell-loading studies were also used to develop a selection system for discriminating between NPD cells and those transduced by retroviral-mediated gene transfer. This selection scheme was based on the fluorescence emission of intact NPD cells, which, when loaded with pyrene-labeled sphingomyelin, was 3- to 5-fold that of normal or transduced cells. As a consequence, the NPD and transduced cells could be efficiently sorted by flow cytometry with a fluorescence-activated cell sorter. In addition, the NPD cells could be selectively killed by photosensitization after irradiation with a long-wavelength UV light. These results should permit direct selection of ASM-expressing cells after retroviral-mediated gene transfer without the need to preselect for a cotransferred marker gene.
authors.affiliation: Division of Medical and Molecular Genetics, Mount Sinai School of Medicine, New York, NY 10029.
authors.forename: M
authors.initials: M
authors.lastname: Suchi
authors.forename: T
authors.initials: T
authors.lastname: Dinur
authors.forename: R J
authors.initials: RJ
authors.lastname: Desnick
authors.forename: S
authors.initials: S
authors.lastname: Gatt
authors.forename: L
authors.initials: L
authors.lastname: Pereira
authors.forename: E
authors.initials: E
authors.lastname: Gilboa
authors.forename: E H
authors.initials: EH
authors.lastname: Schuchman
mesh_terms: D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D002478:Cells, Cultured; D004247:DNA / Q000235:genetics*; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology / Q000528:radiation effects; D005434:Flow Cytometry; D015316:Genetic Therapy; D005822:Genetic Vectors; D006801:Humans; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009542:Niemann-Pick Diseases / Q000201:enzymology* / Q000235:genetics; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D012016:Reference Values; D012867:Skin / Q000201:enzymology / Q000473:pathology / Q000528:radiation effects; D013108:Sphingomyelin Phosphodiesterase / Q000235:genetics* / Q000378:metabolism; D013109:Sphingomyelins / Q000378:metabolism / Q000494:pharmacology; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D011838:Radiation-Sensitizing Agents; D013109:Sphingomyelins; D004247:DNA; D013108:Sphingomyelin Phosphodiesterase