Gene Therapy Program, Department of Pediatrics, and Institute of Human Genetics, University of Minnesota, Minneapolis, MN, USA

Correspondence to: C B Whitley, Gene Therapy Program, University of Minnesota, 420 Delaware Street SE, Minneapolis, MN 55455, USA

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 -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. Gene Therapy (2000) 7, 1875-1883.

mucopolysaccharidosis type I; hematopoietic progenitor cells; vector design; promoters; simplified vectors; bicistronic vectors