NEW YORK – In a pair of studies published in Nature Biotechnology on Thursday, independent research teams from the US and China demonstrated the potential of using circular guide RNAs and a cell's own adenosine deaminase enzymes to do RNA editing.
For the first of these studies, investigators at the University of California, San Diego and Shape Therapeutics shared findings from RNA editing experiments using stable circular RNAs to enlist these endogenous "adenosine deaminases acting on RNA," or ADAR. This circular ADAR-recruiting guide RNA, or cadRNA, strategy was designed to bump up RNA editing efficiency, they explained.
"Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity," senior author Prashant Mali, a bioengineering researcher at UCSD, and his colleagues reported, adding that "targeting via cadRNAs is highly specific at the transcriptome-wide level and, via further engineering to reduce bystander editing, also highly specific at the transcript level."
After a series of reporter assay, deep RNA sequencing, and other experiments to optimize this approach in a human cell line, Mali and his colleagues used cadRNAs and adeno-associated virus, or AAV, vector delivery systems to successfully tweak up to 53 percent of messenger RNAs at a targeted mPCSK9 transcript site in the mouse liver.
Meanwhile, in a mouse model of a rare lysosomal storage condition called mucopolysaccharidosis type I-Hurler syndrome, members of that team reportedly corrected a characteristic nonsense mutation encoded by the IDUA gene between 7 and 17 percent of the time using a cadRNA-based RNA editing approach.
More broadly, the authors noted that "circularization of guide RNAs might also have utility in other transcriptome and genome editing modalities, such as [RNA interference], [antisense oligonucleotides], and guide RNAs in CRISPR-Cas."
In a related Nature Biotechnology study, an independent Peking University and EdiGene team led by Wensheng Wei, a researcher at the Peking University Genome Editing Research Center's Biomedical Pioneering Innovation Center, outlined its own RNA editing approach using covalently closed, circular ADAR-recruiting RNAs (arRNAs), dubbed circ-arRNAs.
"Because editing efficiency depends on the abundance and stability of arRNAs, we evaluate the use of circular RNA, a large class of noncoding RNAs that is highly stable because its covalently closed ring structure protects it from exonucleases," the authors explained.
Their method builds on a "leveraging endogenous ADAR for programmable editing of RNA," or LEAPER, method that Wei and his colleagues originally described in Nature Biotechnology in 2019, using circ-arRNAs and other tweaks to boost RNA editing efficiency and dial down off-target editing events.
In addition to its collaboration with Peking University researchers, the Beijing-based biotechnology company EdiGene is reportedly working on LEAPER-related applications with investigators at the University of Wisconsin at Madison and Peking Union Medical College Hospital.
"LEAPER 2.0 incorporates multiple engineered elements and significantly improves the on-target editing efficiency while reducing the bystander off-target editing events in vitro and in vivo," senior author Wei said in a statement, calling the approach "a powerful and versatile RNA editing technology."
Along with cell line experiments to tweak CTNNB1 or TP53 messenger RNA transcripts, the team turned to LEAPER 2.0 technology to correct a Hurler syndrome-associated point mutation and related metabolic symptoms in a preclinical mouse model of the condition with the help of an AAV vector delivery system.
"[W]e have demonstrated that circ-arRNAs can either be delivered as in vitro-transcribed RNA oligonucleotides or expressed in vivo using an AAV vector," the authors reported, noting that circ-arRNAs "are well suited for delivery by a variety of non-viral vehicles, including lipid nanoparticles and clinical antisense oligonucleotide RNA drugs."