HIGH-LEVEL EXPRESSION AND EXPORT OF BETA-GLUCURONIDASE FROM MURINE MUCOPOLYSACCHARIDOSIS-VII CELLS CORRECTED BY A DOUBLE-COPY RETROVIRUS VECTOR

Retrovirus vectors were constructed to transfer and express the cDNA o f the human lysosomal acid hydrolase beta-glucuronidase (GUSB) under c ontrol of the human GUSB promoter. Expression of the transcription uni t (minigene) was evaluated in a GUSB-negative cell line established fr om a mouse with the lysosomal storage disease mucopolysaccharidosis (M PS) type VII. A vector designed to transfer single copies of the minig ene (N2H beta H) expressed normal levels of GUSB activity in the defic ient cells. GUSB expression was increased to several limes greater tha n normal by inserting the minigene into a double-copy vector (DCH beta H), which places one copy of the transcription unit upstream of the r etrovirus promoter in both the 3' and 5' long terminal repeals (LTRs) of the integrated provirus. The specific activity of GUSB and a contro l normal lysosomal enzyme, alpha-galactosidase (GLA), were higher in n ormal and in vector-corrected cells from confluent cultures than in su bconfluent dividing cells. The ratios of GUSB to GLA were similar at a ll phases of cell growth, but the level of GUSB expression from the do uble copy vector was several-fold higher than from the single copy vec tor. To determine if this effect was controlled by the GUSB promoter a vector was constructed using the thymidine kinase (TK) promoter to dr ive the human GUSB cDNA (NTK beta H). The levels of GUSB in cells corr ected with this vector exhibited the same cell density dependent patte rn as when the GUSB promoter was used, indicating that the variation i n enzymatic activity was not a function of the GUSB promoter. The DCH beta H-infected cells also released higher amounts of GUSB into the cu lture medium than the cells corrected with single-copy vectors. The ab ility of cells transduced with the DCH beta H vectors to express and s ecrete high levels of GUSB may be useful for the delivery of GUSB to M PS VII cells in vivo since the strategy for gene therapy is to use a l imited number of vector-corrected cells as a source of enzyme for the multiple tissues affected in this disease.