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Jason Myers, Jack M Sullivan; Synthetic Nuclease Resistant Enhanced Hammerhead Ribozymes for Nucleic Acid Ocular Therapeutics. Invest. Ophthalmol. Vis. Sci. 2022;63(7):55 – A0028.
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© ARVO (1962-2015); The Authors (2016-present)
We are investigating enhanced hammerhead ribozymes (EhhRzs) that exceed the turnover rate of historical minimal hhRzs (mhhRzs). Small synthetic nuclease resistant EhhRzs are great candidates for intraocular therapeutics. The inner limiting membrane (ILM) which provides a structural boundary between the vitreous and retina allows diffusional entry of small nucleic acids for retinal therapies.
Substrate RHO-266 RNA is 15 nt long with a 5’ FAM and 3’ BHQ1 (IDT); cleavage at CUC 266 results in liberation of quenched fluorescence which is quantified optically in real time. Synthetic EhhRzs are generated by IDT. Cleavage reactions are initiated by mixing EhhRz with substrate RNA in assay buffer (10 mM Tris-HCl, pH 7.5 with 0.5 mM Mg2+) and optically measured at 37°C in a qRT/PCR machine. Nuclease assays are performed in 10% Normal Human Serum (NHS) or recombinant RNase A (100nM) at 37°C. Data are analyzed in Origin.
Chemical modifications (2’-O-Methyl, 2OMe) throughout the EhhRz (except for core nucleotides: G5,G8,G12, A9,A15.1), provides nuclease resistance at 24hrs, but catalytic activity is abolished (1/min vs unmodified 283/min, p=7.30E-12). Locked nucleic acid (LNA) residues incorporated at the 5 and 3’ ends with 2OMe occupied residues in the EhhRz (except for U4, G5,8,12, A9, A15.1, and stem) resulted in catalytic activity (382/min) but lacked nuclease resistance (p=1.11E-6). Antisense oligos designed to protect regions of the EhhRz core demonstrated nuclease protection and provided evidence of susceptibility at U4. An EhhRz incorporating 2OMe, LNA, 2’-Fluor C in the stem, and 2’-Amino U4, maintained strong catalytic activity (112/min) and nuclease resistance at 24hrs.
Chemically-modified EhhRzs that provide nuclease protection while retaining enhanced catalytic activity contribute a useful tool for synthetic nucleic acid ocular therapeutics. These studies incentivize novel chemical biological approaches to further enhance catalytic activity while preserving nuclease insensitivity and extended tissue and cellular half-lives. Synthetic EhhRzs with increased catalytic activity support function under Michaelis-Menten conditions ([S] >> [E]) that allow for robust target mRNA knockdown at lower levels of therapeutic delivery (and toxicity).
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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