Ribozyme rescue of photoreceptor cells in P23H transgenic rats: long-term survival and late-stage therapy.

Ribozyme-directed cleavage of mutant mRNAs appears to be a potentially effective therapeutic measure for dominantly inherited diseases. We previously demonstrated that two ribozymes targeted to the P23H mutation in rhodopsin slow photoreceptor degeneration in transgenic rats for up to 3 months of age when injected before significant degeneration at postnatal day (P) 15. We now have explored whether ribozyme rescue persists at older ages, and whether ribozymes are effective when injected later in the degeneration after significant photoreceptor cell loss. Recombinant adeno-associated virus (rAAV) vectors incorporating a proximal bovine rod opsin promoter were used to transfer either hairpin or hammerhead ribozyme genes to photoreceptors. For the study of long-term survival, rAAV was administered by subretinal injection at P15, and the rats were allowed to live up to 8 months of age. For the study of late-stage gene transfer, rAAV was administered at P30 or P45, when 40-45% of the photoreceptors already had degenerated. Eyes were examined functionally by the electroretinogram and structurally by morphometric analysis. When injected at P15, expression of either ribozyme markedly slowed the rate of photoreceptor degeneration for at least 8 months and resulted in significantly greater electroretinogram amplitudes at least up to P180. When injected at P30 or P45, virtually the same number of photoreceptors survived at P130 as when injected at P15. Ribozyme rescue appears to be a potentially effective, long-term therapy for autosomal dominant retinal degeneration and is highly effective even when the gene transfer is done after significant photoreceptor cell loss.

Ribozyme rescue of photoreceptor cells in a transgenic rat model of autosomal dominant retinitis pigmentosa.

Ribozymes, catalytic RNA molecules that cleave a complementary mRNA sequence, have potential as therapeutics for dominantly inherited disease. Twelve percent of American patients with the blinding disease autosomal dominant retinitis pigmentosa (ADRP) carry a substitution of histidine for proline at codon 23 (P23H) in their rhodopsin gene, resulting in photoreceptor cell death from the synthesis of the abnormal gene product. Ribozymes can discriminate and catalyze the in vitro destruction of P23H mutant mRNAs from a transgenic rat model of ADRP. Here, we demonstrate that in vivo expression of either a hammerhead or hairpin ribozyme in this rat model considerably slows the rate of photoreceptor degeneration for at least three months. Catalytically inactive control ribozymes had less effect on the retinal degeneration. Intracellular production of ribozymes in photoreceptors was achieved by transduction with a recombinant adeno-associated virus (rAAV) incorporating a rod opsin promoter. Ribozyme-directed cleavage of mutant mRNAs, therefore, may be an effective therapy for ADRP and also may be applicable to other inherited diseases.

Ribozyme-targeted destruction of RNA associated with autosomal-dominant retinitis pigmentosa.

PURPOSE: To design ribozymes--catalytic RNA molecules--to cleave the P23H and S334Ter mutant mRNA selectively and to test them in vitro to determine their potential as therapeutic agents in the prevention of autosomal dominant retinitis pigmentosa. METHODS: Synthetic RNA targets were used in cleavage assays to determine the catalytic efficiencies of the ribozymes in vitro. Cleavage products were analyzed by denaturing polyacrylamide gel electrophoresis. Total retinal RNA was also used as a substrate, and opsin mRNA cleavage was assayed by reverse transcription-polymerase chain reaction. RESULTS: All three ribozymes cleaved the mutant target specifically. Substrate cleavage was seen in less than 5 mM magnesium and was detectable after 15 minutes of incubation. The most active ribozyme against the P23H target was the hammerhead (kcat:K(m) [Michaelis-Menton constant] ratio = 5 x 10(7) M/min), then the P23H hairpin ribozyme (kcat:K(m) ratio = 9 x 10(5) M/min) and the S334Ter hammerhead (kcat:K(m) ratio = 8 x 10(5) M/min). No cleavage activity was observed, when wild-type target sequences or inactive control ribozymes were used. The ribozymes bound and specifically digested the intact mutant opsin mRNA in the presence of all normal retinal RNA. CONCLUSIONS: Ribozymes can discriminate between the mutant and wild-type sequences of mRNA associated with autosomal dominant retinitis pigmentosa. The kinetics and specificity of ribozyme cleavage indicate that they should reduce the amount of aberrant rhodopsin in the rod cells and may have potential as therapeutic agents against genetic disease.