title: An allele-specific hammerhead ribozyme gene therapy for a porcine model of autosomal dominant retinitis pigmentosa.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL 32610, USA.
authors.forename: L C
authors.initials: LC
authors.lastname: Shaw
authors.forename: A
authors.initials: A
authors.lastname: Skold
authors.forename: F
authors.initials: F
authors.lastname: Wong
authors.forename: R
authors.initials: R
authors.lastname: Petters
authors.forename: W W
authors.initials: WW
authors.lastname: Hauswirth
authors.forename: A S
authors.initials: AS
authors.lastname: Lewin
mesh_terms: D000483:Alleles; D000818:Animals; D030801:Animals, Genetically Modified; D017931:DNA Primers / Q000737:chemistry; D000229:Dependovirus / Q000235:genetics; D004195:Disease Models, Animal; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D016337:RNA, Catalytic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012174:Retinitis Pigmentosa / Q000235:genetics / Q000628:therapy*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017299:Rod Opsins / Q000235:genetics*; D013552:Swine; D013997:Time Factors; D014162:Transfection
chemical_list: D017931:DNA Primers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D017299:Rod Opsins