Gene therapy of recessive disorders involves the replacement of a defective or missing protein into cells. However, in dominant genetic disorders, such as osteogenesis imperfecta (OI), it is more appropriate to use gene therapy techniques to destroy specific mRNAs to eliminate the dominant negative dysfunction. One approach of message therapy involves the targeted removal of mutant mRNA by ribozyme or antisense technology. We report here the preliminary analysis of ribozymes targeted to mutant type I collagen mRNA in vitro and in cultured cells derived from a patient with OI.

Ribozymes were designed to target a portion of the type I collagen α1 mRNA which surrounds a naturally occurring human mutation (907G→U). This single base substitution generates a GUA cleavage site in the mutant RNA transcript and eliminates a BstN I restriction site. In an in vitro assay, ribozymes cleaved mutant RNA targets whereas normal RNA targets were left intact. For the in cellulo experiments, both active and inactive ribozyme templates were cloned into the pCI.neo and pHβAPr-1-neo mammalian expression vectors, under the control of the CMV and β-actin promoters, respectively. Primary skin fibroblasts were stably transfected with the ribozyme expression vectors by cationic-liposome mediated transfer. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis confirmed the presence of ribozyme transcripts in the stably transfected cells. Quantitative competitive RT-PCR analyses of α1(I) collagen normalized to β-actin expression levels, revealed that the level of mutant type I collagen α1 mRNA was decreased (approximately 50%) in the cells containing active ribozyme in comparison to untransfected cells.

Further studies are in progress to assess the levels of mutant and normal type I collagen protein in the stably transfected cells. Additionally, transient transfection assays are being undertaken to determine the effect of modifications (length and mismatches) to the binding arms on the cleavage specificity in cellulo.