June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Structural Modelling Predicts Variability in Efficacy of Lead Candidate Hammerhead Ribozyme Between Different Species Rhodopsin mRNA
Author Affiliations & Notes
  • Alexandria J Trujillo
    Research Service, VA Western NY Healthcare System, Buffalo, New York, United States
    Pharmacology and Toxicology, University at Buffalo, Buffalo, New York, United States
  • Beau R Froebel
    Ophthalmology (Ross Eye Institute), University at Buffalo, 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, Neuroscience Program, University at Buffalo, Buffalo, New York, United States
  • Footnotes
    Commercial Relationships   Alexandria Trujillo, None; Beau Froebel, None; Jack Sullivan, None
  • 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, 4493. doi:
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    • Get Citation

      Alexandria J Trujillo, Beau R Froebel, Jack M Sullivan; Structural Modelling Predicts Variability in Efficacy of Lead Candidate Hammerhead Ribozyme Between Different Species Rhodopsin mRNA. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4493.

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

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Abstract

Purpose : Hammerhead ribozymes (hhRz) cleave target mRNAs at ‘NUH’ (N= any nucleotide H= U, A, C) sites. When developed for therapeutic purposes the target site must be in a region of the mRNA that is accessible in a stable structure in vivo. Our lab has designed a hhRz that cleaves the 725nt region of human Rhodopsin mRNA (hRHO), as a therapeutic for Autosomal Dominant Retinitis Pigmentosa (adRP.) Conventional methods to bring a therapeutic to clinical trial generally require efficacy and toxicity studies conducted in non-human animals. Rat, mouse, pig, and dog are commonly used as adRP model organisms in therapeutic screening. This study was conducted to determine the potential impact that use of non-human animals would have on evaluating a ribozyme that targets hRHO mRNA, and to further characterize and optimize our 725 GUC↓ hhRz.

Methods : RHO mRNA sequences for Human and 15 non-human animals (relevant to adRP/evolutionary relations) were folded at the secondary level using SFold, MFold, and RNAStructure (OligoWalk) algorithms. These algorithms predict structure based on single stranded accessibility by Boltzmann weighting, minimal folding energy, and local free energy, respectively. The product of these output vectors determine the Multiparameter Prediction of RNA accessibility (MPPRNA) and were graphed and area under the curve (AUC) was calculated.

Results : For all animals (n=15) MPP values and AUC varied from human regardless of sequence homology in both regions. One-way ANOVA demonstrated MPP values were significantly different (p<0.05) between all species, and 2-way independent t-tests showed AUC values were significantly different (p<0.05) from human for the majority of animal RHO mRNAs tested (n=9, 60%).

Conclusions : Primary mRNA sequence or protein homology is insufficient information to predict mRNA accessibility and efficacy of post-transcriptional gene silencing agents such as hhRzs. Algorithms that predict mRNA folding provide an expedited analysis for structural similarity to the human mRNA target as a proxy for cleavage efficacy. Because of the predicted structural variations between even evolutionarily close organisms, in a single gene that encodes highly homologous proteins, development studies for PTGS agents should be conducted with human samples or mRNAs derived from human genes in transgenic animals.

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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