Design of vectors for efficient expression of human purine nucleoside phosphorylase in skin fibroblasts from enzyme-deficient humans.

Abstract

Purine nucleoside phosphorylase (PNP; purine-nucleoside orthophosphate ribosyltransferase, EC 2.4.2.1) deficiency is an inherited disorder associated with a severe immune defect that is fatal. Enzyme replacement therapy is an attractive approach to treatment of this disease. To this aim we constructed retroviral vectors containing a human PNP cDNA and a selectable gene encoding neomycin phosphotransferase. PNP expression was controlled by either the early promoter from simian virus 40, the immediate early promoter from human cytomegalovirus, or the retroviral promoter. Cultured-skin fibroblasts from two unrelated PNP. deficient patients that were infected with these vectors expressed mean PNP activities of0.03, 0.74, and 5.9 pmol/hr per mg of protein, respectively. The latter infectants had PNP activities eight times the level of 0.74 ,umol/hr per mg of protein observed in normal skin fibroblasts, enabling rapid metabolism of exogenous deoxyguanosine, the cytotoxic metabolite that accumulates in the plasma of PNP-deficient patients. These experiments indicate that viral long terminal repeat was the strongest promoter for expression of PNP and suggest the potential of human skin fibroblasts as vehicles for therapeutic gene expression.

Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

“…Nevertheless, mast cell numbers can be restored by transplantation of normal bone marrow (Kitamura et al, 1978) (Figure 2), which correlated with functional receptor expression (Figure 3). These observations extend previous studies (Osborne and Miller, 1988;Hock et al, 1989;Palmer et al, 1989) to confirm the greater efficiency of the retroviral LTR over a number of internal promoter elements. However, the applicability of internal promoters seems also to depend on the nature of the inserted sequence, Hock et al, 1989;Keller and Wagner, 1989).…”

Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.

“…Virus titers measured on NIH 3T3 cells ranged from 106 to 8 x 106 colony-forming units (cfu) per ml. As noted in previous studies, the titers were generally 10-fold lower on normal diploid fibroblast strains from either humans or rats (6,9,12,13). …”

Long-term expression of human adenosine deaminase in vascular smooth muscle cells of rats: a model for gene therapy.

“…Vascular smooth muscle cells offer the same advantage in terms of proximity to the circulation; indeed, in cases of vascular injury where the endothelium ofthe vessel is not completely reestablished, smooth muscle cells form a pseudoendothelium that is in direct contact with the blood (6). In addition, while endothelial cells exist only as a monolayer, smooth muscle cells can form multiple layers and provide an estimated 10-fold increase in potential target cells, which may be important where relatively large numbers of genetically modified cells are necessary for therapy (7,8).…”