Co-expression of MGMT(P140K) and alpha-L-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction.
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BACKGROUND: Systemic in vivo gene therapy has resulted in widespread correction in animal models when treated at birth. However, limited improvement was observed in postnatally treated animals with mainly targeting to the liver and bone marrow. It has been shown that an O(6)-methylguanine-DNA-methyltransferase variant (MGMT(P140K)) mediated in vivo selection of transduced hematopoietic stem cells (HSC) in animals. METHODS: We investigated the feasibility of MGMT(P140K)-mediated selection in primary hepatocytes from a mouse model of mucopolysaccharidosis type I (MPS I) in vitro using lentiviral vectors. RESULTS: We found that multiple cycles of O(6)-benzylguanine (BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment at a dosage effective for ex vivo HSC selection led to a two-fold increase of MGMT-expressing primary hepatocytes under culture conditions with minimum cell expansion. This enrichment level was comparable to that obtained after selection at a hepatic maximal tolerated dose of BCNU. Similar levels of increase were observed regardless of initial transduction frequency, or the position of MGMT (upstream or downstream of internal ribosome entry site) in the vector constructs. In addition, we found that elongation factor 1alpha promoter was superior to the long-terminal repeat promoter from spleen focus-forming virus with regard to transgene expression in primary hepatocytes. Moreover, the levels of therapeutic transgene expression in transduced, enzyme-deficient hepatocytes directly correlated with the doses of BCNU, leading to metabolic correction in transduced hepatocytes and metabolic cross-correction in neighbouring non-transduced MPS I cells. CONCLUSIONS: These results demonstrate that MGMT(P140K) expression confers successful protection/selection in primary hepatocytes, and provide 'proof of concept' to the prospect of MGMT(P140K)-mediated co-selection for hepatocytes and HSC using BG/BCNU treatment. (+info)
Targeting of the CNS in MPS-IH using a nonviral transferrin-alpha-L-iduronidase fusion gene product.
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Uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts and osteoblasts.
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To examine the uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts from a patient with mucopolysaccharidosis I (MPS I) and its effect on the cleavage of accumulated substrates, we performed enzymological, Western blotting, immunocytochemical and morphological studies. Laronidase was incorporated into the MPS I cells dose-dependently mainly via mannose 6-phosphate (M6P) receptors. Then the incorporated enzyme was transported to lysosomes and processed to the mature form, the pathological changes of the cells being improved. Furthermore, we compared the uptake of laronidase by cultured mouse osteoblasts with that by cultured mouse fibroblasts. The enzyme was incorporated into the cultured mouse osteoblasts mainly via M6P receptors, although mannose (Man) receptors were partially involved in the uptake of the enzyme, as in the cultured fibroblasts. But the uptake by the former was apparently lower than that by the latter. The administration of a high dose of the enzyme or development of a recombinant alpha-L-iduronidase containing many M6P residues is required for further improvement of enzyme replacement therapy for skeletal disorders caused by MPS I. (+info)
Aptamer-based endocytosis of a lysosomal enzyme.
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alpha-L-iduronidase in normal and mucopolysaccharidosis-type-I human skin fibroblasts.
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alpha-L-Iduronidase synthesis and maturation were analysed in fibroblasts from normal controls and from alpha-L-iduronidase-deficient mucopolysaccharidosis-type-I (MPS-I) patients. Fibroblasts were radiolabelled with [3H]leucine and alpha-L-iduronidase was isolated from cell lysates or culture medium by monoclonal-antibody affinity chromatography. Pulse-chase labelling of normal control fibroblasts showed that alpha-L-iduronidase was synthesized as an 81 kDa precursor and processed within 24 h via intermediates of 76 kDa and 70 kDa to a 69 kDa species. The incorporation of radiolabel into alpha-L-iduronidase in fibroblasts from three of four MPS-I patients was at levels that were either very low or undetectable. Fibroblasts from one MPS-I patient, however, exhibited levels of incorporation of radiolabelled amino acid into alpha-L-iduronidase similar to those shown by normal control fibroblasts, despite having undetectable alpha-L-iduronidase enzyme activity. The maturation of alpha-L-iduronidase in fibroblasts from this patient was delayed compared with normal controls and showed accumulation of the 76 kDa intermediate, as well as the major 69 kDa, form of the enzyme. (+info)