Optimizing Enhanced Hammerhead Ribozymes for Retinal Nucleic Acid Therapeutics

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Many mutations in the human rod opsin (hRHO) and peripherin genes cause autosomal dominant retinitis pigmentosa (adRP) and macular degenerations. The folded mRNAs are targets for mutation-independent hammerhead ribozyme (hhRz) gene therapy. Our long-range goal is to translate effective hhRz therapeutics for hRHO adRP and other genes into human clinical trials. We have discovered a potent form of hhRz, which we call Enhanced-hhRz (EhhRz) therapeutics. All or most known mutations in a given dominant disease gene could be treated with a mutation-independent EhhRz strategy that pairs knockdown (KD) of mutant (and WT) mRNA with reconstitution (RECON) of WT protein with expression of a cleavage-resistant mRNA. The current stage of development of EhhRzs (against hRHO) lead to kinetic turnover rates greater than 2-log orders improved over historical minimal hhRzs (mhhRz); one mhhRz partially rescued mutant RHO retinal degeneration in rodent models. EhhRzs function under substrate (target) excess conditions and physiological levels of cofactor Mg2+, which are optimum for intracellular KD therapeutics. If this exciting kinetic potential can be harnessed for the photoreceptor, retinal, or ocular cells then there is potential for nucleic acid drugs (injectable EhhRzs) as therapeutics without vector-based gene therapy, for diverse ocular diseases and inherited retinal degenerations. Recent clinical success with intravitreal injection of antisense agents incentivizes this development. The objective is to optimize EhhRzs for intracellular performance in cultured human cells and mouse photoreceptor cells that are “humanized” for RHO mRNAs (same targets as in clinical trials). The central hypothesis is that optimization of EhhRz kinetic performance and delivery will maximize target mRNA/protein KD; optimization requires quantitation of biophysical variables that underlie target: EhhRz interaction, rational and/or evolutionary engineering of RNA structure-related function, and identification of the optimum delivery materials and approach. Our rationale is that EhhRzs can be optimized through synthetic chemistry and/or natural modifications, while retaining or enhancing kinetic function, to support robust intracellular KD. Treatment occurs through vector-mediated delivery (AAV) of expression constructs, or direct delivery of synthetic EhhRz therapeutics (injectables). To test the central hypothesis the Specific Aims are: Aim 1. Optimize current EhhRzs at lead hRHO target sites (266 CUC, 725 GUC, 1362 GUC) for rate enhancement, cellular stability, target-colocalization, and knockdown performance. Aim 2. Optimize EhhRz Knockdown strategy for: (i) rescue (efficacy) of retinal degeneration and (ii) lack of toxicity with AAV-EhhRz gene therapy and injectable synthetic EhhRzs in transgenic mouse models humanized for RHO. Innovation occurs through optimization of EhhRz design, and optimization of strategies for therapeutic delivery in a humanized adRP model. Significance- The study optimizes EhhRz function and delivery as an initial step toward a KD/RECON rescue strategy for dominant photoreceptor degenerations.