Nucleic Acids Research, 2000, Vol. 28, No. 20 4013-4020
© 2000 Oxford University Press
Hammerhead ribozymes selectively suppress mutant type I collagen mRNA in osteogenesis imperfecta fibroblasts
Section on Connective Tissue Disorders, Heritable Disorders Branch, NICHD, National Institutes of Health, Bethesda, MD 20892, USA
Ribozymes are a promising agent for the gene therapy of dominant negative genetic disorders by allele-specific mRNA suppression. To test allele-specific mRNA suppression in cells, we used fibroblasts from a patient with osteogenesis imperfecta (OI). These cells contain a mutation in one 
1(I) collagen allele which both causes the skeletal disorder and generates a novel ribozyme cleavage site. In a preliminary in vitro assay, ribozymes cleaved mutant RNA substrate whereas normal substrate was left intact. For the studies in cell culture we generated cell lines stably expressing active (AR) and inactive (IR) ribozymes targeted to mutant 1(I) collagen mRNA. Quantitative competitive RT–PCR analyses of type I collagen mRNA, normalized to ß-actin expression levels, revealed that the level of mutant 1(I) collagen mRNA was significantly decreased by 
50% in cells expressing AR. Normal 1(I) collagen mRNA showed no significant reduction when AR or IR was expressed from the pHßAPr-1-neo vector and a small (10–20%) but significant reduction when either ribozyme was expressed from the pCI.neo vector. In clonal lines derived from cells expressing AR the level of ribozyme expression correlated with the extent of reduction in the mutant:normal 1(I) mRNA ratio, ranging from 0.33 to 0.96. Stable expression of active ribozyme did not affect cell viability, as assessed by growth rates. Ribozyme cleavage of mutant mRNA results in a reduction in mutant type I collagen protein, as demonstrated by SDS–urea–PAGE. This is the first report of ribozymes causing specific suppression of an endogenous mutant mRNA in cells derived from a patient with a dominant negative genetic disorder.
* To whom correspondence should be addressed at: Building 10, Room 9S241, 10 Center Drive, MSC 1830, Bethesda, MD 20892-1830, USA. Tel: +1 301 496 6683; Fax: +1 301 402 0234; Email: oidoc@helix.nih.gov Present address: Paul A. Dawson, Department of Physiology and Pharmacology, University of Queensland, Brisbane, Australia
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