Variables and Strategies in Development of Therapeutic Post-Transcriptional Gene Silencing Agents
Figure 5
Predicted minimal free energy folding structures of human rod opsin and Bestrophin-1 mRNAs. GeneBank accession numbers for human rod opsin mRNA (NM000539.2) and Bestrophin-1 mRNA (NM004183) are indicated. (a) Human rod opsin mRNA (1–1820 nt) was folded in silico with RNA-Fold. The minimal free energy structure is shown. Note the dense secondary structure with only rare single-stranded annealing platforms of any substantial size. Also shown are the locations of human missense mutations that cause autosomal dominant retinitis pigmentosa and that generate new hhRz cleavage sites. These are mostly buried in secondary structure, where they would be poor targets for a mutation-specific (MSpe) approach to gene therapy as the PTGS agent would have limited capacity to anneal and cleave only the mutant target mRNA. (b) Human bestrophin-1 mRNA (1–2000 nt) was folded with RNA-fold and the MFE structure is shown. Again, secondary structure is dense with rare single-stranded annealing platforms larger than 10 nt. The locations of human mutations that cause autosomal dominant best macular dystrophy and that generate new hhRz cleavage sites for a mutation-specific strategy are shown (MSpe). Most are buried in dense secondary structure, where they would be expected to be inaccessible to annealing of a PTGS agent. Also shown are some mutations located in regions of WT cleavage sites, where they would permit a mutation-selective (MSel) approach to PTGS gene therapy.
