Molecular Therapeutic Approaches for Treatment of Autosomal Dominant RP (ADRP) in a Canine T4R Rhodopsin Model

Abstract

Abstract: : Purpose: The recently characterized dominant canine T4R rhodopsin (RHO) mutation leads to a slowly progressive RP-like disease that offers the opportunity to develop a therapy for human ADRP in a human-sized eye. Our aims were to design hammerhead and X-motif ribozymes and a small interfering RNA (siRNA) for wild type and T4R RHO mRNAs and to test them in tissue culture cells as a preamble to testing in the dog. Methods: Several ribozymes were designed, including an allele-specific T4R X -motif and two hammerhead ribozyme targeting both the T4R mutant and wild type RHO mRNA. Ribozyme activity was initially determined by reaction time course and multiple turnover kinetic analysis using short RNA targets. A cell line stably expressing the canine T4R RHO was isolated using Zeocin selection. Transient transfection of HEK 293 cells with RHO and ribozyme expression plasmids was performed using different molar ratios of target to ribozyme plasmid (1:4; 1:6). Evaluation of mRNA levels was by quantitative RT-PCR. We also designed a siRNA for the first exon of the RHO gene, and cloned it into an expression vector driven by H1 RNA polymerase III promoter. Results:The allele specific T4R RHO X-motif ribozyme was tested in vitro but was significantly less catalytically active than either hammerhead ribozyme. Kcat's for hammerhead ribozymes Rz 397 and Rz 448 were 1.66min-1 and 14.8 min-1 respectively on short RNA targets Transient co-transfection of 293 cells with wild type RHO target and either Rz 397 or Rz 448 resulted in approximately 70% reduction of RHO mRNA at 3 days post transfection. Transfection of a 293 cell line stably expressing RHO with the siRNA vector led to reduction of RHO mRNA by more than 50%. Conclusions: Allele independent hammerhead ribozymes Rz 397 and Rz 448 are more catalytically active than a T4R X-motif ribozyme in vitro. Significant cellular reduction of RHO mRNA was achieved by both ribozymes and by the siRNA. Either or both may be useful in a gene therapy strategy to treat ADRP.

Keywords: gene transfer/gene therapy • retinitis • gene mapping