High level expression and export of β-glucuronidase from murine mucopolysaccharidosis VII cells corrected by a double-copy retrovirus vector

Retrovirus vectors were constructed to transfer and express the cDNA of the human lysosomal acid hydrolase β-glucuronidase (GUSB) under control of the human GUSB promoter. Expression of the transcription unit (minigene) was evaluated in a GUSB-negative cell line established from a mouse with the lysosomal storage disease mucopolysaccharidosis (MPS) type VII. A vector designed to transfer single copies of the minigene (N2HβH) expressed normal levels of GUSB activity in the deficient cells. GUSB expression was increased to several times greater than normal by inserting the minigene into a double-copy vector (DCHβH), which places one copy of the transcription unit upstream of the retrovirus promoter in both the 3' and 5' long terminal repeats (LTRs) of the integrated provirus. The specific activity of GUSB and a control normal lysosomal enzyme, α-galactosidase (GLA), were higher in normal and in vector-corrected cells from confluent cultures than in subconfluent dividing cells. The ratios of GUSB to GLA were similar at all phases of cell growth, but the level of GUSB expression from the double copy vector was several-fold higher than from the single copy vector. To determine if this effect was controlled by the GUSB promoter, a vector was constructed using the thymidine kinase (TK) promoter to drive the human GUSB cDNA (NTKβH). The levels of GUSB in cells corrected with this vector exhibited the same cell density dependent pattern as when the GUSB promoter was used, indicating that the variation in enzymatic activity was not a function of the GUSB promoter. The DCHβH-infected cells also released higher amounts of GUSB into the culture medium than the cells corrected with single-copy vectors. The ability of cells transduced with the DCHβH vectors to express and secrete high levels of GUSB may be useful for the delivery of GUSB to MPS VII cells in vivo since the strategy for gene therapy is to use a limited number of vector-corrected cells as a source of enzyme for the multiple tissues affected in this disease.