William W. Hauswirth

Ribozymes Directed Against Messenger RNAs Associated With Autosomal Dominant Retinitis Pigmentosa

Ribozymes are RNA enzymes that can be modified to cleave almost any target RNA. Because of this versatility, they are useful in digesting transcripts of dominant mutant genes leading to retinal disease, including ADRP. One approach to gene therapy for this disease would be to reduce the expression of the mutant opsin that interferes with the accumulation of active rhodopsin and is responsible for a substantial fraction of ADRP. We have developed ribozymes designed to cleave mutant mRNA molecules leading to several opsin missense and nonsense mutations that cause retinal degeneration in human and in animal models. These include the P23H, G90D, S334 termination, and the P347S mutations. Both hairpin and hammerhead ribozymes have been tested. The efficiency of cleavage depends on the nucleotide triplet at the cleavage site and its context in the mRNA. All ribozymes tested are capable of cleaving short synthetic targets, longer cloned targets and full length opsin mRNA containing the mutant target sequence. In no case has cleavage of the wild-type opsin been detected. For these reasons, ribozymes hold promise as sequence-specific tools for gene therapy for ADRP. Animal tests of these ribozymes are underway using Adeno-Associated Virus vectors to deliver DNA copies of the ribozymes to affected transgenic animals.