UC San Diego

ABSTRACT OF THE THESIS

RNA Editing via Recruitment of Endogenous ADARs using Circular Antisense Guide RNAs

By

James Jen Yen

Master of Science in Bioengineering

University of California San Diego, 2021

Professor Prashant Mali, Chair

Genetic disorders collectively chronically affect 1 in 17 individuals in the world today. Currently, many of the treatments that exist for such disorders are palliative and only treat the symptoms, not the underlying cause. The small amount of approved curative treatments that do exist utilize permanent genome editing tools that carry inherent risks of permanent off-target modifications. The aim of this thesis is to develop a platform for the safe and effective repair of genetic disorders using A-I RNA editing. It has been recently shown that delivery of long antisense guide RNAs can recruit endogenous adenosine deaminase acting on RNA (ADAR) enzymes to induce RNA editing in vitro. Importantly however, this approach is unable to induce RNA editing in vivo; we hypothesized this to be a result of the short half-life of linear guide RNAs resulting from vulnerability to exonuclease attack. By engineering and delivering highly stable circular guide RNAs via AAV8, we were able to induce robust RNA editing in mice livers: we observed 53% editing in the 3’UTR of the mPCSK9 transcript in C57BL/6J mice and 12% correction of a nonsense mutation in the IDUA-W392X mouse model for type mucopolysaccharidosis type I-Hurler (MPS I-H) syndrome. Furthermore, we were able to reduce the bystander editing profile of target transcripts by engineering loop secondary structures strategically placed throughout our circular antisense guide RNAs. Altogether, our platform paves the way for safe transcript-specific RNA editing for use in gene therapy.