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eSymposia | RNA Editing and Modifications: From Biology to Therapy


Stereopure oligonucleotides support efficient ADAR-mediated RNA editing in non-human primates


Sep 30, 2020 12:00am ‐ Sep 30, 2020 12:00am

Description

Stereopure oligonucleotides support efficient ADAR-mediated RNA editing in non-human primates Prashant Monian1, Chikdu Shivalila1, Luciano Apponi1, Bijay Bhattarai1, Adam Bezigian1, David Boulay1, Michael Byrne1, Arindom Chatterjee1, Jigar Desai1, Frank Favaloro1, Jack Godfrey1, Andrew Hoss1, Naoki Iwamoto1, Jayakanthan Kumarasamy1, Pachamuthu Kandasamy1, Tomomi Kawamoto1, Anthony Lamattina1, Richard Looby1, Genliang Lu1, Jake Metterville1, Ronelle Murphy1, Snehlata Tripathi1, Stephany Standley1, Hailin Yang1, Yuan Yin1, Hui Yu1, Chandra Vargeese1,* 1Wave Life Sciences, *Corresponding author Recruiting endogenous RNA-editing enzymes using chemically modified oligonucleotides holds great promise for treating human disease. The ADAR (adenosine deaminases acting on RNA) family of enzymes catalyze adenine (A) to inosine (I) changes in coding and non-coding sequences throughout the transcriptome. Because inosine (I) is read as guanine (G) by the translational machinery, oligonucleotide-directed ADAR-mediated RNA editing has the potential to revert myriad disease-causing mutations through a variety of molecular mechanisms, including protein restoration, gene silencing, altered splicing, and protein alteration. PRISMTM, Wave Life Sciences’ proprietary discovery and drug development platform, enables us to develop stereopure oligonucleotides—those in which the chiral configuration of backbone linkages (e.g., Rp or Sp) are precisely controlled at each position—to target genetically defined diseases. We report the application of PRISM to develop stereopure oligonucleotides that support efficient ADAR-mediated RNA editing. We first explore the impact of oligonucleotide chemistries (modifications to the sugar and backbone), backbone stereochemistry and other aspects of design (e.g., length, mismatches) on the activity of RNA-editing oligonucleotides in an extensive structure-activity relationship (SAR) analysis using a dual-luciferase reporter system in vitro. We apply learnings from this SAR analysis to develop optimized, stereopure editing oligonucleotides: we demonstrate that stereopure oligonucleotides can direct endogenous ADAR enzymes to edit endogenous transcripts in vitro, and that oligonucleotides optimized based on our SAR analysis were more effective than stereorandom oligonucleotides. Stereopure oligonucleotides can elicit efficient site-directed RNA editing in primary human hepatocytes when delivered under gymnotic (i.e., free uptake) conditions or with an N-acetylgalactosamine (GalNAc) conjugate. We demonstrate that our design strategy is applicable to multiple sequences by showing ADAR-mediated editing of five endogenous transcripts, and we demonstrate that it is effective in multiple cell types in vitro, including primary human hepatocytes, primary human fibroblasts, and bronchial epithelial cells. Finally, we show that the most effective oligonucleotides identified in our in vitro studies support up to 50% editing of the b-actin transcript in vivo in the liver of non-human primates. Taken together, these preclinical investigations lay the foundations for development of PRISM-generated stereopure RNA-editing therapeutics with potential to treat human genetic disease. This work was funded by Wave Life Sciences.

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