An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.

Published online

Journal Article

PURPOSE: To develop a hammerhead ribozyme-based gene therapy for a porcine model of autosomal dominant retinitis pigmentosa (ADRP). METHODS: Hammerhead ribozymes were developed and assayed in vitro against RNA targets homologous to the opsin P347S mutants found in a transgenic porcine model and in humans. Both cloned and synthetic RNA oligonucleotide versions of ribozymes and targets were tested under multiple-turnover conditions using oligonucleotide RNA targets. Digestion of full-length P347S mRNA from porcine retina was performed. RESULTS: The porcine P347S hammerhead ribozyme was specific for the opsin P347S sequence. Multiple-turnover analysis yielded the following kinetic parameters: Vmax=7.3+/-0.5 nM/min, Km=2.1+/-0.6 mM, and kcat=1.5+/-0.4 min-1. The human P347S hammerhead ribozyme was substantially less active (~10,000 fold). CONCLUSIONS: We have developed a hammerhead ribozyme to use as a model for gene therapy of autosomal dominant retinitis pigmentosa in a transgenic porcine model. Based on kinetic characterization of this ribozyme compared to others used for gene therapy, this should be an effective reagent RNA. The allele specific ribozyme we tested for the human sequence, however, is not likely to be useful for gene therapy indicating that an alternative approach is necessary.

Full Text

Duke Authors

Cited Authors

  • Shaw, LC; Skold, A; Wong, F; Petters, R; Hauswirth, WW; Lewin, AS

Published Date

  • January 26, 2001

Published In

Volume / Issue

  • 7 /

Start / End Page

  • 6 - 13

PubMed ID

  • 11172137

Pubmed Central ID

  • 11172137

Electronic International Standard Serial Number (EISSN)

  • 1090-0535


  • eng

Conference Location

  • United States