Abstract
Purpose :
There are two strategies of nucleic acid therapeutics for autosomal dominant retinal or ocular degenerations: mutation-dependent and mutation-independent strategies. In mutation-dependent strategies, the mutation in the mRNA sequence may create the opportunity to attack, cleave, and ablate only the mutant mRNA while leaving the wild type (WT) mRNA intact. In mutation-independent strategies, a cleavage site is chosen in a highly accessible region of the target mRNA. This region is present in almost all mutant mRNAs and WT mRNA. Additionally, the therapeutic must be paired with an allelic variant WT expression construct, which reconstitutes WT protein expression. The human rhodopsin gene (hRHO) offers a robust case of many disease-causing mutations (>150), mostly single nt changes. The capacity to cleave mRNA depends upon the local accessibility around the target binding site, which is a critical bottleneck in any nucleic acid therapeutic modality (antisense, ribozyme, shRNA). Here, we evaluate the critical variable of local target mRNA accessibility of multiple mutant hRHO mRNAs vs. WT hRHO mRNA.
Methods :
Multiparameter prediction of RNA accessibility (mppRNA), developed in the Sullivan lab, was used to quantify 2-D accessibility in a set of mutant hRHO mRNAs (N=35) in the region of the mutation (where the therapeutic would anneal) vs. the 2D accessibility in the identical region of the WT hRHO mRNA. A standard 23 nt platform was used with the single nt mutation centered in the platform. mppRNA utilizes vector outputs from MFold, SFold, and Oligowalk algorithms input to a bioinformatics model. Statistical analysis was performed in Origin.
Results :
26 of 35 mutations (74%) did not significantly change local accessibility vs. WT (p>0.05). 4 of 35 mutations (11%) significantly decreased local accessibility vs. WT (p<0.05). 5 of 35 mutations (14%) significantly increased local accessibility vs. WT (p<0.05). For ribozymes, only 1 mutation creates new cleavage sites.
Conclusions :
This bioinformatics approach demonstrates that the mutation-specific approach is unlikely to be broadly useful to treat dominant mutations. Even where it might be applicable, each therapeutic agent is a separate development for relatively small numbers of patients with an orphan disease. A mutation-independent strategy is more rational and cost-effective.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.