Restoration of normal lysosomal function in mucopolysaccharidosis type VII cells by retroviral vector-mediated gene transfer.

Abstract

Retroviral vectors were constructed containing a rat .8-glucuronidase cDNA driven by heterologous pro-

In utero transplantation of fetal liver cells in the mucopolysaccharidosis type VII mouse results in low-level chimerism, but overexpression of β-glucuronidase can delay onset of clinical signs

“…The TG donor cells produce supernormal levels of GUSB and can be distinguished from C3H tissues, which have low levels of normal GUSB enzyme activity compared with other strains. 22,23,29 The transgene is constitutively expressed; thus, the cells mimic vector-transduced cells that are overexpressing the gene of interest, 20 without the disadvantage of down-regulation of the vector. If only a small number of cells can be transplanted or there is a low rate of engraftment, cells overexpressing the gene of interest may be able to compensate for the difference.…”

“…Overexpression of a normal lysosomal enzyme from transgenic cells transplanted postnatally has been shown to reverse lesions in another storage disease model, 19 and retroviral vectors can overexpress GUSB in transduced hematopoietic cells. 12,20,21 Constitutive expression of GUSB was studied by using FLCs from an allogeneic TG mouse strain that expresses very high levels of human GUSB against the MPS VII background. The in utero transplantation properties of these allogeneic cells were compared with retroviral marking studies of syngeneic cell transplants.…”

A single-base-pair deletion in the beta-glucuronidase gene accounts for the phenotype of murine mucopolysaccharidosis type VII.

“…Stable transformants were selected in medium containing G418 at 200 pg/ml (Gentecin, GIBCO). Ten days after transfection, cells expressing t3-glucuronidase activity were detected by a histochemical stain using naphthol AS-BI 3-D-glucuronide (Sigma) and pararosaniline hydrochloride (13).…”

“…Genomic DNA was isolated from the spleen of a wild-type B6.C-H-2bml/By mouse and from the spleen of a gusmPs/gusmPs mouse obtained from the B6.C-H-2bml/ByBir-gusmPs/+ mutant strain (1). The DNAs were partially digested with Mbo I, and fragments ranging in size from [9][10][11][12][13][14][15][16][17][18][19][20] Ci/mmol; 1 Ci = 37 GBq) in 50 mM Tris, pH 7.5/5 mM MgCl2/1 mM dithiothreitol/0.5 mM each of dATP, dTTP, and dGTP for 1 hr at 37°C. The uniformly radiolabeled DNA was phenol-chloroform extracted, ethanol precipitated, and digested with EcoRI.…”

Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

“…The canine MPS VII myoblasts were used as a model system to determine if vector-mediated gene transfer into myoblasts could be used to correct a genetic deficiency in differentiated myotubes. The MPS VII myoblasts were infected with the N2 vector, containing only the neo gene, or with the NTK-BGEO-1 vector, which has been shown to express normal levels of P-glucuronidase activity in other canine and human MPS VII cells (15). The vector-infected myoblasts proliferated, fused, and differentiated into myotubes in the presence of G418 (Fig.…”