The Long and Short of Designing Allele-Specific Hammerhead Ribozymes: Gene Therapy for Rhodopsin-Linked ADRP

Abstract

Abstract: : Purpose: More than a hundred point mutations in the rhodopsin gene are known cause autosomal dominant retinitis pigmentosa (ADRP), a progressive rod dystrophy. We have designed therapeutic hammerhead ribozymes for two common disease-causing alleles of rhodopsin, P23H and G90D. Since neither of these allelic variants creates an optimal hammerhead ribozyme cleavage site, our goal was to identify suitable NUX cleavage triplets and design mutation-specific hammerhead ribozymes. Methods: Sequence data was used to design several related hammerhead ribozymes to target either the human P23H rod opsin mRNA or murine G90D opsin mRNA. In vitro cleavage time course analyses were performed using short synthetic RNA substrates containing either the mutant or wild-type rod opsin mRNA sequences to determine the selectivity (allele specificity) and catalytic efficiency of the ribozymes. Modifications were made in the consensus catalytic core, the central conserved stem-loop, and both hybridizing arms, to enhance the allele specificity and kinetic properties of the human P23H ribozymes. To improve the catalytic properties of the G90D ribozymes, we assayed several NUX cleavage triplets around the mutation site. Hammerhead ribozymes that selectively cleaved the mutant RNA substrate but not the normal sequence were subjected to further kinetic analysis. Ribozymes demonstrating favorable kcat values were cloned and packaged into recombinant AAV virus particles. The AAV vectored human P23H ribozymes are currently being tested in cell culture. The therapeutic potential of the G90D ribozymes is presently being assayed in transgenic mice. Results: For both the human P23H and G90D transgene, hammerheads ribozymes with asymmetrically designed hybridizing arms demonstrated the highest degree of allele discrimination and yielded the most catalytically favorable properties. Conclusions: The sequence context of the targeted cleavage triplet, as well as the length of the hybridizing arms, greatly impact the allele specificity of hammerhead ribozymes.

Keywords: gene transfer/gene therapy