Self-cleaving multimeric hammerhead ribozymes abolish mutant collagen transcripts in cellulo and exhibit activity in vivo

Severe forms of osteogenesis imperfecta are produced by dominant negative mutations in type I collagen. These mutations allow the inclusion of aberrant protein in type I collagen molecules. Therefore, therapy for this disorder must eliminate mutant transcripts prior to translation of mutant protein. This can be achieved by hammerhead ribozymes: small catalytic RNA molecules that can be targeted to any RNA molecule containing a putative nucleotide triplet cleavage site. Osteogenesis imperfecta was modeled in cellulo by stable expression of a COL1A1 minigene (pMG155) in MC3T3-E1 osteoblasts. This minigene expresses a truncated COL1A1 in which exons 5 and 47 are fused, creating a ribozyme cleavage site. A monomeric ribozyme containing a 3' self-cleaving site (Rz547) was designed to target this cleavage site. A multimeric ribozyme (M8Rz547) containing 8 tandemly arranged copies of the monomer was then produced. The self-cleaving multimeric ribozyme was designed to increase the titer and stability, and therefore the effective activity, of the ribozyme under physiological conditions. Expression of both Rz547 and M8Rz547 are driven by the COL1A1 promoter. Rz547 and M8Rz547 have previously been shown to cleave the COL1A1 target sequence, and to show self cleavage, when tested in vitro. The activities of the monomeric and multimeric ribozymes were compared in cellulo by stable co-expression of either Rz547 or M8Rz547 and the COL1A1 minigene target in MC3T3-E1 osteoblasts. Both Rz547 and M8Rz547 exhibited self-cleavage activity in cellulo. The titer of ribozyme was significantly increased in the cells expressing the multimeric M8Rz547 compared to the monomeric Rz547. Ribozymes were expressed in the nucleus and transported to the cytoplasm. The monomeric Rz547 produced a modest reduction of the target RNA and protein, compared to the dramatic elimination of target transcript and protein in the cells expressing the multimeric M8Rz547 ribozyme. The osteoblasts co-expressing M8Rz547 and the COL1A1 minigene target showed a reversion to a normal collagen phenotype, demonstrated by increased collagen fibril diameter and increased collagen deposition To examine the activity of the multimeric ribozyme in vivo, a transgenic mouse expressing the M8Rz547 has been generated. Self cleavage of the multimeric ribozyme was seen in tail RNA from these mice, demonstrating that the multimer is active in vivo. These experiments demonstrate the increased activity that multimerization confers to ribozymes targeting specific transcript sequences, and demonstrates the utility that multimeric ribozymes may have as a gene therapy tool.