title: Expression of the human glucocerebrosidase and arylsulfatase A genes in murine and patient primary fibroblasts transduced by an adeno-associated virus vector.
abstract: We have constructed two recombinant adeno-associated virus (AAV) vectors (pJJ-3GC and pJJ-3ASA) which contained either the human glucocerebrosidase (GC) or arylsulfatase A (ASA) cDNA under the control of an SV40 promoter. These plasmids were co-transfected to 293 cells with a helper plasmid containing trans-acting AAV genes required for packaging the vectors. The two recombinant vectors successfully infected murine and patient fibroblasts. The human glucocerebrosidase and arylsulfatase A genes were expressed at high levels in the cells as determined by Western blot analysis, enzyme assay and immunochemical staining. GC enzyme activity in Gaucher patient fibroblasts (GM-0877) infected by AAV-GC was 15-fold higher than in non-infected cells. ASA enzyme activity in MLD 557g cells infected by AAV-ASA was up to 500-fold higher than in the metachromatic leukodystrophy (MLD) control cells. Southern blotting results showed that the vector integrated 1-2 copies of pJJ-3GC and ASA in the targeted cell genome. These two vectors will be useful in studying AAV-mediated transfer of the GC and ASA genes in cells and animals.
authors.affiliation: Department of Human Genetics, University of Pittsburgh, PA, USA.
authors.forename: J F
authors.initials: JF
authors.lastname: Wei
authors.forename: F S
authors.initials: FS
authors.lastname: Wei
authors.forename: R J
authors.initials: RJ
authors.lastname: Samulski
authors.forename: J A
authors.initials: JA
authors.lastname: Barranger
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002553:Cerebroside-Sulfatase / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D005347:Fibroblasts; D015870:Gene Expression; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D005962:Glucosylceramidase / Q000235:genetics*; D006801:Humans; D051379:Mice; D011401:Promoter Regions, Genetic; D013539:Simian virus 40 / Q000235:genetics; D014161:Transduction, Genetic
chemical_list: D018076:DNA, Complementary; D002553:Cerebroside-Sulfatase; D005962:Glucosylceramidase