Abstract
PURPOSE: The mutation-independent strategy for hammerhead ribozyme (hhRz) or RNA interference (RNAi)-based gene therapeutics to treat autosomal dominant diseases is predicated on the hypothesis that a single therapeutic would equivalently suppress all/most of the diverse mutant mRNAs in patients with the disease phenotype. However, the hypothesis has not been formally tested. We address this through a comprehensive bioinformatics study of how mutations affect target mRNA structure accessibility for a single lead hhRz therapeutic (725GUC↓), designed against human rod rhodopsin mRNA (hRHO), for patients with hRHO mutations that cause autosomal dominant retinitis pigmentosa. METHODS: A total of 199 in silico coding region mutations (missense, nonsense, insert, deletion, indel) were made in hRHO mRNA based on Human Gene Mutation Database and Database of Single Nucleotide Polymorphisms. Each mRNA was folded with MFold, SFold, and OligoWalk algorithms and subjected to a bioinformatics model called multiparameter prediction of RNA accessibility. Predicted accessibility of each mutant over both a broad local region and the explicit lead ribozyme annealing site were compared quantitatively to wild-type hRHO mRNA. RESULTS: Accessibility of the 725GUC↓ site is sensitive to some mutations. For single nucleotide missense mutations, proximity of the mutation to the hhRz annealing site increases the impact on predicted accessibility, but some distant mutations also influence accessibility. CONCLUSIONS: A mutation-independent strategy appears viable in this specific context but certain mutations could significantly influence ribozyme or RNAi efficacy through impact on accessibility at the target annealing site/region. This possibility must be considered in applications of this gene therapy strategy.