Specific hammerhead ribozymes reduce synthesis of cation-independent mannose 6-phosphate receptor mRNA and protein

Storage diseases because of lysosomal enzyme deficiencies may be treated by the transplantation of cells that secrete the enzyme which is deficient in patients. One can expect that increasing the amount of secreted enzymes will improve the therapy efficacy. Secretion of lysosomal enzymes can be enhanced by reducing the mannose 6-phosphate receptor involved in the lysosomal sorting of newly synthesized lysosomal enzymes. For this purpose, we have constructed hammerhead ribozymes targeting the mRNA of the large murine mannose 6-phosphate receptor (M6PR300). In vitro ribozymes cleave M6PR300 RNA fragments efficiently with cleavage rates of 69–93% after 3 h of incubation. Ribozymes were cloned into an expression vector in which they are integrated into the VaI adenovirus RNA to increase stability and in which they are transcribed from an RNA polymerase III promoter. These plasmids were transiently transfected into BHK cells to investigate in vivo activity. Two ribozymes reduce efficiently the levels of murine M6PR300 mRNA in transient transfection experiments to 42–45%. This correlates with the reduction of M6PR300 biosynthesis, which is reduced also to 37% of normal. We can also demonstrate that the reduction in M6PR300 is sufficient to increase a lysosomal enzyme secretion.