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Jack M Sullivan, Jason Myers, Alexandria Trujillo, Mark Christian Butler, Zahra Fayazi, Jibin A. Punnoose, Kenneth A. Halvorsen; Facilitated Hammerhead Ribozymes- A New Therapeutic Modality for Inherited Retinal Degenerations. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3412.
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© ARVO (1962-2015); The Authors (2016-present)
Ribozyme technology as a candidate gene therapeutic was supplanted (RNAi and CRISPR) before critical knowledge emerged on structures and mechanisms that enhance RNA catalysis. We investigated RNA structure/function relationships in a novel Facilitated-hammerhead ribozyme (F-hhRz) which cleaves human rod rhodopsin (hRHO) mRNA with high catalytic activity under substrate excess and cellular MgCl2 levels. F-hhRz is a novel therapeutic for RHO adRP/Stargardt/dry AMD and can be targeted to arbitrary mRNAs.
Ribozyme and hRHO RNAs were transcribed (T7) from templates or synthesized. In vitro hhRz cleavage assays used full length (1532nt), fragment (~500nt) mRNAs, or miniature 15-mer substrates with 5’FAM and 3’BHQ1 (cleavage yields fluorescence). Isothermal titration calorimetry (ITC) assessed binding thermodynamics of noncleavable and cleavable substrates.
The Facilitator (50nt), when placed 3’ of a minimal hhRz, enhances rapid target cleavage under substrate excess at cellular Mg2+ levels against all tested substrates. Inverse RNA folding identified sequences with the same 2D structure but variant 3D structures. Facilitators (n=10) enhance (up to >4X to 115/min p=8.2E-10) or suppress activity consistent with 3D structure. Minimal hhRzs embedded in protective scaffolds have 1-2 log order slower rates. Rational mutagenesis identified Facilitator regions important for function, and a truncated form (37 nts) with similar catalytic enhancement (p<0.05). ITC demonstrates that: 1) Facilitator must be in cis to the hhRz to impact binding (no measureable trans interaction), 2) Facilitator promotes a marked (110-fold) shift in Kd of substrate binding (1.1mM for 266-hhRz vs 10nM for 266-F-hhRz) suggesting strong and specific interaction(s), and 3) these interactions are not observed if substrate can be cleaved (suggesting high enzymatic turnover).
Minimal hhRzs cap at 1-2/min rate and use only two of four possible RNA chemistry rate enhancement mechanisms; protein RNaseA uses all four. The impressive rate enhancement of the F-hhRz (> 100/min) infers use of more than two catalytic enhancement strategies. Further rational investigation is yielding F-hhRzs that compete with protein level catalytic function as site-specific designer RNA endonucleases for gene therapy. Our discovery revitalizes ribozyme therapeutics as highly specific and potent RNA drugs for IRDs.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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