Retroviral vector design studies toward hematopoietic stem cell gene therapy for mucopolysaccharidosis type I

To optimize a gene transfer system for hematopoietic stem cell gene therapy of patients with mucopolysaccharidosis (MPS) type 1, 10 retroviral vectors were constructed to express the human alpha -L-iduronidase (IDUA) cDNA. Th ese vectors were designed to evaluate the potential effects of specific pro moters, the addition of selectable markers, and the use of multiple promote rs versus an infernal ribosome entry site for expression of IDUA and select able maker genes. The effect of vector design was investigated in primary p atient fibroblasts (F-MPS) or murine fibroblast cell lines; while overall c omparison of transgene expression was determined in patients' peripheral bl ood lymphocytes (PBLMPS) and CD34(+) progenitors (PBPCMPS). We observed tha t the human PGK promoter introduced the highest IDUA activity per 1% relati ve transgene frequency in F-MPS. Use of the same promoter to separately reg ulate both the therapeutic gene and a drug-resistance gene resulted in decr eased expression of the unselected gene. Go-selection using bicistronic vec tors not only increased the number of transductants, but also elevated tran sgene expression under selective pressure in transgene-positive progenitors . Bicistronic vector LP1CD overcame down-regulation and practically introdu ced the highest IDUA level in unselected PBLMPS and an intermediate level i n PBPCMPS. These studies provide a better understanding of factors contribu ting to efficient gene expression in hematopoietic cells.