Osteogenesis imperfecta (OI) is a paradigm for understanding the molecular basis of heritable diseases of connective tissues. Two fundamentally different mechanisms for defective collagen formation underlie the disease: mutations which encode a defective protein and act in a dominant negative manner, and mutations that reduce normal collagen production by inactivating one collagen allele. This laboratory has focused on the latter group (null mutations) typically found in patients with type I OI and heritable osteoporosis. The applicant has shown that fibroblasts from patients with OI containing certain splice donor mutations, premature stop codons or frame-shift mutations which induce premature stop codons yield transcripts that are retained in the nucleus. Molecular and confocal in situ hybridization studies performed in the previous grant period indicate that the complexities of collagen mRNA processing are not modeled by current laboratory techniques. Specifically, cells transfected with genes containing the same mutations found in OI fibroblasts do not yield the same outcome. Whereas both systems sequester RNA transcripts with a retained intron, the levels of the mutant transcript in the nucleus differ dramatically. RNA transcripts with a premature stop codon are restricted to the nuclear compartment in OI cells, but escape to the cytoplasm when the same mutation is transfected into normal cells. This renewal application proposes to more fully contrast the differences between the two cell types and identify different steps in the pathway of RNA processing that are blocked when RNA transcripts harbor a premature stop codon or retained intron. The second objective of the renewal is to utilize the emerging understanding of RNA processing to develop new strategies to inactivate a dominant negative mutation in the COL1A1 gene. This is a fundamental need for somatic gene therapy of OI. A rapid assay to assess the effectiveness of ribozymes and U1 snRNA constructs to reduce expression of a collagen-like transcript will be developed as a testing ground for reagents which might have applicability to discriminate a one nucleotide difference between two collagen transcripts. Both approaches utilize recent advances in promoters to drive expression of RNAs with features designed to optimize their interaction with the target transcript. Armed with these reagents, a murine model of a glycine point mutation will be made to test whether the mutant allele can be specifically down-regulated when the U1 snRNA or ribozymes are directed to a glycine point mutation and polymorphic regions in cis with the mutation.
