Osteogenesis Imperfecta (O.I.) is a heritable generalized disorder of connective tissue caused by a dominant mutation in either α chain of type I collagen. As the selective reduction of the mutated allele mRNA level should represent a good strategy for the therapy of the disease, we focused our preliminary studies on the in vitro activity of four hammerhead ribozymes targeted against naturally occurring mutations found in three unrelated patients.
For each patient, a cDNA fragment from both normal and mutated allele has been cloned in pGEM3z vector and mRNA was generated by in vitro transcription. Two pmoles of either mutated or normal mRNA were incubated with the specific ribozyme in a final volume of 10 μl. Increasing amounts of ribozyme (Rz/substrate: 1:1; 5:1; 25:1, 125:1), were mixed with the substrate and the incubation was carried out at both 37°C and 50°C for 25′, 50′ and 75′. Samples were separated on a 10% denaturing polyacrylamide gel, stained with ethidium bromide and photographed. After determining the intensity of the bands by densitometric scanning, we calculated the ratio between the intensity of the undigested substrate and that of the digested product. For the two cleavage sites “GUU” and“UUC”, we further analyzed the in vitro ribozyme activity by increasing the length of the mRNA up to 900.
