Co-expression of MGMTP140K and α- L-iduronidase in primary hepatocytes from mucopolysaccharidosis type I mice enables efficient selection with metabolic correction.

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 O⁶-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 O⁶-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 1α 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. Copyright © 2007 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]