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RNA editing involves the direct alteration of RNA transcripts through the addition, deletion, or substitution of nucleotides. This offers the ability to post-transcriptionally modify proteins by recoding transcripts at the RNA level. The primary focus of therapeutic RNA editing has been on converting adenosine to inosine (A-to-I) in a programmable manner using adenosine deaminases acting on RNA (ADARs). This technology enables the correction of disease-causing G-to-A mutations, restoration of nonsense mutations, and modulation of pre-mRNA splicing and other regulatory motifs that rely on adenosines.
Understanding the biology of ADAR enzymes - there are 3 ADAR genes in humans that encode 5 isoforms. ADAR1 and ADAR2 are catalytically active, while ADAR3 lacks activity but may regulate editing. ADAR1 is ubiquitously expressed and critical for regulating innate immunity. ADAR2 expression is more limited but plays a key role in recoding specific transcripts.
The structure and sequence preferences of ADAR1 and ADAR2 impact their editing efficiency and specificity. This knowledge can inform the design of guide RNAs (gRNAs) that preferentially recruit one isoform over the other. Editing is influenced by RNA accessibility, with structured regions being more challenging to edit. The RBP landscape also impacts editing, as ADAR competes with other dsRBPs. Nuclear import and export regulate access to nuclear vs cytosolic substrates. This impacts the subcellular localization requirements for therapeutic gRNAs.
gRNA design considerations - gRNAs can be DNA-encoded or chemically synthesized antisense oligos. Design parameters differ based on the delivery method. For DNA-encoded gRNAs, circularization improves stability and persistence of editing. Subcellular localization can be controlled through promoters and RBP binding sites. Knowledge of ADAR structure informs approaches to improve gRNA efficiency and specificity. Addition of structures to the gRNA:mRNA duplex can reduce off-target editing. Shorter chemically synthesized gRNAs simplify manufacturing but length needs to be balanced with efficiency. Chemical modifications also impact editing and can be optimized.
Obvious applications are correcting missense and nonsense mutations, with 7,000 known pathogenic G-to-A mutations. Editing splice sites, start codons, miRNA binding sites can modulate splicing, expression levels and open other opportunities. Recoding transcripts can alter protein function, like enzyme active sites or PPI interfaces. This enables new possibilities beyond antibodies.
Excellent specificity is required to avoid unintended transcriptome changes. Methods to measure on- and off-target editing events are critical. The impact of off-target events needs careful evaluation. Not all edits will alter protein function so the consequences require case-by-case analysis. The delivery method impacts biodistribution and cell targeting. AAV and ASO delivery have distinct advantages and challenges that influence clinical development. Quantifying RNA editing and protein restoration in patients can inform dose selection but is limited by accessibility of target tissues. Preclinical data can guide starting dose. Careful trial design is needed to learn about RNA editing, protein and phenotypic outcomes in early clinical studies.
RNA editing is poised to impact medicine, but hurdles related to delivery, specificity, manufacturing, and clinical validation still remain. Overcoming these challenges will require cross-disciplinary collaboration between fields like RNA biology, oligo chemistry, viral engineering, and drug development. If successful, the tunability and reversibility of RNA editing could enable therapies for many diseases beyond what is achievable with current genetic medicines.
