title: Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I.
abstract: To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type I, 10 retroviral vectors were constructed to express the human alpha-L-iduronidase (IDUA) cDNA. These vectors were designed to evaluate the potential effects of specific promoters, the addition of selectable markers, and the use of multiple promoters versus an internal ribosome entry site for expression of IDUA and selectable maker genes. The effect of vector design was investigated in primary patient fibroblasts (F(MPS)) or murine fibroblast cell lines; while overall comparison of transgene expression was determined in patients' peripheral blood lymphocytes (PBL(MPS)) and CD34+ progenitors (PBPC(MPS)). We observed that the human PGK promoter introduced the highest IDUA activity per 1% relative transgene frequency in F(MPS). Use of the same promoter to separately regulate both the therapeutic gene and a drug-resistance gene resulted in decreased expression of the unselected gene. Co-selection using bicistronic vectors not only increased the number of transductants, but also elevated transgene expression under selective pressure in transgene-positive progenitors. Bicistronic vector LP1CD overcame down-regulation and practically introduced the highest IDUA level in unselected PBL(MPS) and an intermediate level in PBPC(MPS). These studies provide a better understanding of factors contributing to efficient gene expression in hematopoietic cells.
authors: Department of Pediatrics, and Institute of Human Genetics, University of Minnesota, Minneapolis 55455, USA.
authors: D
authors: D
authors: Pan
authors: E
authors: E
authors: Aronovich
authors: R S
authors: RS
authors: McIvor
authors: C B
authors: CB
authors: Whitley
mesh_terms: D000818:Animals; D002460:Cell Line; D004351:Drug Resistance / Q000235:genetics; D005347:Fibroblasts / Q000201:enzymology; D015870:Gene Expression; D005818:Genetic Engineering; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D006801:Humans; D007068:Iduronidase / Q000032:analysis / Q000235:genetics*; D008727:Methotrexate; D051379:Mice; D008059:Mucopolysaccharidosis I / Q000628:therapy*; D010735:Phosphoglycerate Kinase / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012190:Retroviridae / Q000235:genetics; D014162:Transfection
chemical_list: D010735:Phosphoglycerate Kinase; D007068:Iduronidase; D008727:Methotrexate