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Jack M Sullivan, Beau R Froebel; Bioinformatics Analysis of Utility of the Mutation-Independent Approach for Hammerhead Ribozyme Therapy of Autosomal Dominant RP due to RHO Mutations . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5463.
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© ARVO (1962-2015); The Authors (2016-present)
A hammerhead ribozyme (hhRz) (725 GUC↓), in supportive RNA scaffolds, targets human rod opsin mRNA (hRHO), demonstrates cleavage in vitro and in cellulo, and exerts substantial cellular target knockdown. This hhRz is a candidate gene therapeutic for all known hRHO mutations causing autosomal dominant retinitis pigmentosa (adRP), in a mutation-independent strategy, where both intrinsic mutant and wild type (WT) mRNA are reduced, and WT protein is reconstituted through transcription of an allelic variant WT mRNA that cannot be cleaved. Success depends upon equivalent hhRz efficacy against various adRP mutant mRNAs. We investigated the impact of local and remote hRHO mutations on mRNA accessibility around the 725 cleavage site.
A total of 81 individual mutations in full length hRHO mRNA (1-1532 nt) were made in silico. Each mutant and WT RHO mRNA were folded at the secondary structural level using three algorithms: MFold, SFold, and OligoWalk. Vector outputs as maps of mRNA accessibility were combined into a single map of access probabilty along the mRNA. For each mRNA accessibility in the local region (640-764 nt) around the hhRz 725 cleavage site and explicitly at the annealing site (718-732 nt) were measured by focused integration in the access map.
A total of 59 single nt missense mutations were made within 200 nt of the 725 cleavage site. Additional single nt mutations (P23H, C187Y, P347X) outside this region were made, as well as small or large insertions/deletions, and small indels. Qualitatively, the region of accessibility around the 725 cleavage site was grossly maintained in most single nt mutations. Many single nt mutations exerted little effect on either measured local or site accessibility while some exerted changes that increased or decreased access probability. Accessibiliy change has an exponential distribution relative to the distance of the mutation from the cleavage site (76 nt constant, p=3.6E-18). Impact of insertions/deletions was dependent on their size and distance from the cleavage site.
A hRHO adRP mutation is more likely to have an impact on utility of the mutation-independent strategy of hhRz gene therapy if it occurs proximate to the target cleavage site or exerts gross mRNA folding perturbation. This outcome is likely to follow for other hhRz cleavage sites in hRHO or other targets.
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