Ribozyme-mediated gene therapy for autosomal dominant retinal degeneration

Ribozymes targeted to a point mutation (histidine for proline substitution at position 23) in rhodopsin were evaluated as therapeutic agents for photoreceptor degeneration in an animal model of autosomal dominant retinitis pigmentosa (ADRP). Recombinant adeno-associated virus (AAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. AAV was administered by subretinal injection at postnatal day (P) 15 to rats expressing a rod opsin transgene with a proline-to-histidine substitution in exon 1 at codon 23 of the rhodopsin gene (P23H). In vivo levels of normal and mutant RNAs were measured by allele-specific RT-PCR at P60. Eyes were examined by histopathology and morphometric analysis, as well as by ERG at approximately P60, P75 or P90. Expression of either a hammerhead or hairpin ribozyme markedly slowed the rate of photoreceptor degeneration for at least 3 months as determined by outer nuclear layer thickness as well as by inner and outer segment length. Catalytically inactive control ribozymes had a significantly smaller effect on the retinal degeneration. Minimal rescue effects were seen in retinas injected with either AAV containing a gene for green fluorescent protein rather than the ribozyme gene or buffer only. The level of mutant opsin RNA relative to wild-type RNA was also reproducibly lower in ribozyme-treated retina. Finally, ERG comparisons of ribozyme-treated versus control eyes demonstrated functional rescue. We conclude that ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapy for autosomal dominant retinal degeneration.