Design and Validation of Therapeutic Hammerhead Ribozymes for Autosomal Dominant Diseases

Hammerhead ribozymes are small, catalytic RNAs that can be designed to effectively inhibit gene expression in an allele-specific manner. It is the high level of sequence discrimination, coupled with the minimal cleavage-site requirements of hammerhead ribozymes, that makes these catalytic RNAs so amenable for use as therapeutic agents for autosomal dominant diseases. Here, we present a detailed set of protocols for the design and validation of hammerhead ribozymes for the treatment of autosomal dominant disease, with specific examples of hammerhead ribozymes targeted against human P23H rod opsin mRNA, a major cause of dominant retinitis pigmentosa.