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Jason Myers, Zahra Fayazi, Mark Christian Butler, Jack M Sullivan; Hammerhead ribozyme in a tRNA scaffold: Effects of RNA processing and anti-sense flank length on cleavage in-vivo. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4496.
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Embed a therapeutic hammerhead ribozyme (hhRz) within a human tRNA-Lys3 scaffold and screen modifications to achieve and optimize a gene therapy agent for Stargardt/dry AMD/adRP by suppression of human rod rhodopsin (hRHO).
tRNA hhRz processing and mRNA were assayed by endpoint RT/PCR and real time RT/PCR. hRHO knockdown by various 725 hhRzs and varying anti-sense (AS) flank lengths was assayed by RT/PCR, Alexa-647-1D4 IHC , and a reporter from a bicistronic construct (phRHO-IRES-SuperNova). HhRz kinetic model of Stage-Zimmermann and Uhlenbeck (1998) and RNAstructure were used for modelling.
tRNAs are transcribed by RNA Pol III, but addition of an upstream H1 promoter/enhancer increases tRNA levels substantially. Fractionation and RNA isolation from H1 tRNA hhRz transfected cells with a 3’ CCA lead to loss of hhRz during processing, while lack of 3’ CCA allows nuclear export where the tRNA hhRz reaches a cytoplasmic steady-state (SS) concentration which modulates collision frequency with hRHO mRNA. When SS is reached cleavage efficiency depends on the hhRz enzyme kinetics which are influenced by the length of the hhRz AS flanks. Kinetic modelling of our initial hhRz with 11 nt 5’ and 7 nt 3’ AS flanks predicted reaction kinetics with a lack of binding specificity (Kbinding = 1.74E-17 M) and product inhibition (product leaving rates of 6.67E-4 min-1 and 2.1 min-1). In transfected cells this tRNA hhRz led to an increase in hRHO levels (mRNA, protein) compared to tRNA scaffold alone (p=0.01009). Decreasing 725 AS flanks to 7/7, 6/6 and 5/5 dropped RHO levels significantly (>50%) compared to Scaffold (7/7 p=0.02072, 6/6 p=1.42E-5, 5/5 p=1.16E-4). Our new lead candidate H1 725 6/6 -CCA had a binding affinity of 0.49 nM and leaving rates of 5 min-1 and 17.8 min -1 which provides specificity without product inhibition. Using Supernova as a reporter for hhRz cleavage, H1 tRNA 725 6/6 –CCA suppressed fluorescence levels by 72% compared to scaffold (p=3.25E-52).
A therapeutic 725 hhRz driven by the H1 promoter/enhancer with 6nt/6nt AS flanks suppresses levels of membrane bound hRHO protein by 50% in transient transfection experiments. This tRNA hhRz delivered by AAV to photoreceptors has potential to rescue light-induced damage of the ABCA4/RDH8 double knockout mouse (Stargardt, dAMD model) and intrinsic retinal degeneration in our humanized adRP hRHO mouse.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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