June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Cleavage of Human Rhodopsin mRNA and Protein Knockdown using Lead HHRz-725, a Candidate Therapeutic for Autosomal Dominant Retinitis Pigmentosa.
Author Affiliations & Notes
  • Zahra Fayazi
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology (Ross Eye Institute), University at Buffalo-SUNY, Buffalo, New York, United States
  • Mark Christian Butler
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology (Ross Eye Institute), University at Buffalo-SUNY, Buffalo, New York, United States
  • Jack M Sullivan
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Ophthalmology (Ross Eye Institute), Pharmacology/Toxicology, Physiology/Biophysics and Program in Neuroscience, University at Buffalo-SUNY, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Zahra Fayazi, None; Mark Butler, None; Jack Sullivan, Research Foundation of SUNY (P)
  • Footnotes
    Support  NIH/NEI R01EY013433 (JMS); Veterans Administration Merit Award 1I01 BX000669 (JMS); SUNY Health Now Award (JMS); Research to Prevent Blindness Unrestricted Award (Univ. Buffalo)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 4494. doi:
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      Zahra Fayazi, Mark Christian Butler, Jack M Sullivan; Cleavage of Human Rhodopsin mRNA and Protein Knockdown using Lead HHRz-725, a Candidate Therapeutic for Autosomal Dominant Retinitis Pigmentosa.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4494.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Lead hammerhead ribozyme (hhRz)-725 embedded in an engineered anticodon domain of the small human tRNA-Lys3 scaffold has potential as a therapeutic agent for autosomal dominant retinitis pigmentosa (adRP). Following demonstrated improved catalytic activity of hhRz-725 in vitro, we tested activity in cell culture. Advancement in characterization places us one step closer to testing this post transcriptional gene silencing (PTGS) agent in an adRP animal model.

Methods : tRNA-Ribozymes were cloned into a plasmid with T7 promoter to drive in vitro transcription. Full length human RHO (hRHO) including 5' UTR and part of 3'UTR was cloned into plasmids driven by T7 promoter for in vitro and CMV for expression into HEK-293S cells. In vitro cleavage reactions used T7 RNA polymerase for co-transcription and cleavage of hhRz and hRHO RNAs with analysis by PAGE. Real time PCR for hRHO was performed using primers spanning exon1 and 2 and an internal probe containing a fluorescent dye (FAM) at the 5' and a quenching dye (BHQ1) at the 3' end and b-actin as endogenous control and analyzed by comparative CT method (ΔΔCT method).

Results : We studied cleavage activity of hhRz-725 in vitro and cell culture on short and full length versions of hRHO mRNA. Since we found hhRz-725 has superior cleavage activity in the context of a smaller tRNA scaffold than prior adenoviral VAI scaffold, we tested it with no scaffold and four different constructs containing a smaller stem-II in hhRz-725 (Mini-725) but showed no enzymatic improvement. Looking at the combination of hhRz-725 and hhRz-266 and hhRz-1364 with hRHO the catalytic function showed independent cleavage activity for each hhRz. Quantification of hRHO mRNA by real time RT/PCR and protein using 1D4 antibody to hRHO in cells transfected with VAI-hhRz-725 showed ~40% knockdown both in mRNA (p=1.58865E-4) and protein level (p=1.08485E-6). Also tRNA-hhRz-725 showed ~40 % hRHO mRNA (p=0.01688) knockdown. While less specific PTGS agent shRNA at the (725 GUC↓) showed more than 90% (p=2.92915E-7) RHO mRNA knockdown by real time RT/PCR.

Conclusions : We are optimizing our lead hhRz-725 in the human tRNA scaffold and showed hRHO mRNA cleavage in vitro and also knockdown of hRHO mRNA and protein in cell culture. We are now in the process of advancing the ribozyme for testing in a humanized mouse model of adRP.

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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