Enhanced intracellular availability and survival of hammerhead ribozymes increases target ablation in a cellular model of osteogenesis imperfecta.

Antisense hammerhead ribozymes have the capability to cleave complementary RNA in a sequence-dependent manner. In osteogenesis imperfecta, a genetic disorder of connective tissue, mutant collagen type I has been shown to participate in but not sustain formation of the triple helix. Selective ablation of mutant collagen gene transcript could potentially remove the mutant gene product and reverse the dominant-negative effect exerted by the abnormal protein. In earlier studies we showed that the hammerhead ribozyme Col1A1Rz547 selectively cleaved a mutant Col1A1 gene transcript in a murine calvarial osteoblast cell line. In order to test the possible therapeutic efficacy of this approach, a dramatic downregulation of the mutant transcript must be achieved, a function directly related to high steady-state level of intracellular ribozyme. We report significantly enhanced expression of Col1A1Rz547 by vaccinia T7 polymerase following infection with an attenuated T7-pol vaccinia virus as shown both by the intracellular level of the ribozyme and the cleavage of the mutant Col1A1 gene transcript. We also describe the engineering of a multimeric ribozyme construct comprising eight subunits, which can self-cleave to monomers. These studies suggest the potential use of multimeric ribozymes expressed by a vaccinia-based system in the therapy of a variety of disorders.

Novel approach to the molecular diagnosis of Marfan syndrome: application to sporadic cases and in prenatal diagnosis.

Marfan syndrome is an autosomal dominant disorder affecting the skeletal, ocular, and cardiovascular systems. Defects in the gene that encodes fibrillin-1 (FBN1), the main structural component of the elastin-associated microfibrils, are responsible for the disorder. Molecular diagnosis in families with Marfan syndrome can be undertaken by using intragenic FBN1 gene markers to identify and track the disease allele. However, in sporadic cases, which constitute up to 30% of the total, DNA-based diagnosis cannot be performed using linked markers but rather requires the identification of the specific FBN1 gene mutation. Due to the size and complexity of the FBN1 gene, identification of a causative Marfan syndrome mutation is not a trivial undertaking. Herein, we describe a comprehensive approach to the molecular diagnosis of Marfan syndrome that relies on the direct analysis of the FBN1 gene at the cDNA level and detects both coding sequence mutations and those leading to exon-skipping, which are often missed by analysis at the genomic DNA level. The ability to consistently determine the specific FBN1 gene mutation responsible for a particular case of Marfan syndrome allows both prenatal and pre-implantation diagnosis, even in sporadic instances of the disease.

Delivery of a hammerhead ribozyme specifically downregulates mutant type I collagen mRNA in a murine model of osteogenesis imperfecta.

Osteogenesis imperfecta (OI) is a systemic heritable disorder of connective tissue, caused by a mutation in one of the genes for type I collagen, whose cardinal manifestation is bone fragility. Several studies have identified two molecular mechanisms of collagen type I defects. In chain exclusion, the mutant chain is not incorporated into the collagen triple helix, whereas in chain nonexclusion, it is. The dominant-negative effect of nonexcluded mutations must be taken into account in all strategies aimed at correcting the collagen defects in individuals affected with moderate or several OI. Herein, we describe the application of hammerhead ribozymes to selectively target the mutant minigene transcript expressed in a murine calvarial osteoblast cell line. Active and control inactive ribozymes were tested in vitro on both mutant and normal targets and in the minigene-expressing cell line. Active ribozyme cleaved its target with high efficiency and specificity in both a time-dependent and dose-dependent manner. After delivery of a ribozyme expression construct, intracellular ribozyme was detected, along with a relative reduction in mutant transcript level.

Split-hand/split-foot malformation is caused by mutations in the p63 gene on 3q27.

Split-hand/split-foot malformation (SHFM), a limb malformation involving the central rays of the autopod and presenting with syndactyly, median clefts of the hands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals, and metatarsals, is phenotypically analogous to the naturally occurring murine Dactylaplasia mutant (Dac). Results of recent studies have shown that, in heterozygous Dac embryos, the central segment of the apical ectodermal ridge (AER) degenerates, leaving the anterior and posterior segments intact; this finding suggests that localized failure of ridge maintenance activity is the fundamental developmental defect in Dac and, by inference, in SHFM. Results of gene-targeting studies have demonstrated that p63, a homologue of the cell-cycle regulator TP53, plays a critically important role in regulation of the formation and differentiation of the AER. Two missense mutations, 724A-->G, which predicts amino acid substitution K194E, and 982T-->C, which predicts amino acid substitution R280C, were identified in exons 5 and 7, respectively, of the p63 gene in two families with SHFM. Two additional mutations (279R-->H and 304R-->Q) were identified in families with EEC (ectrodactyly, ectodermal dysplasia, and facial cleft) syndrome. All four mutations are found in exons that fall within the DNA-binding domain of p63. The two amino acids mutated in the families with SHFM appear to be primarily involved in maintenance of the overall structure of the domain, in contrast to the p63 mutations responsible for EEC syndrome, which reside in amino acid residues that directly interact with the DNA.