RNA Editing as a Therapeutic Approach for Retinal Gene Therapy Requiring Long Coding Sequences

Abstract

RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.

Genome-Editing Strategies for Treating Human Retinal Degenerations

“…The majority of RNA editors utilise the deaminase domain from the human adenosine deaminase acting on RNA type 2 (ADAR2 DD ) which converts adenosine to inosine predominantly in protein-coding regions of pre-mRNA in its native role. The newly formed inosine is then interpreted as guanosine when translated by the ribosomal machinery 51 .…”

“…Rational mutagenesis of ADAR2 DD to generate cytidine deaminase activity and subsequent fusion to dCas13b from Riemerella anatipestifer (dRanCas13b) yielded RESCUE (RNA Editing for Specific C-to-U Exchange) 53 . Together, REPAIR and RESCUE provide scope for correcting up to 61% of pathogenic point mutations within mRNAs 51 . Furthermore, C-terminal truncation of dPspCas13b and dRanCas13b in REPAIR and RESCUE, respectively, affords active RNA editors that are small enough to be packaged into single AAVs.…”

Genome editing technologies: CRISPR, LEAPER, RESTORE, ARCUT, SATI, and RESCUE

“…However, some studies favor ADAR because it does not have immune-stimulating effects and does not interfere with the function of endogenous ADAR proteins. When engineered ADARs for RNA editing are examined, apart from the A-to-I editors mentioned so far, there are also C-to-U editors, although they are not often mentioned (Fry et al, 2020[ 35 ]). A deaminase has been developed that allows the regulation of cytosine and uracil in RNA by mutagenesis of ADAR2.…”

“…RNA regulation (RESCUE) for specific C-to-U exchange is the RNA regulation method created by serial mutagenesis (Fry et al, 2020[ 35 ]) of fused ADAR deaminase domains (ADARDD) to dRanCas13b (Cox et al, 2017[ 27 ]). The need for new techniques is due to deficiencies in existing technologies.…”

An overview of applications of CRISPR-Cas technologies in biomedical engineering

“…Similar to CRISPR-Cas9 which present base editing activity to revert an SNP without inducing a dsDNA break [56], RNA base editing for A-to-I edit [57] and C-to-U edit [58] were proven to be achieved by fusion of dCas13 with ADAR. These systems are used to make changes at RNA level as well as to treat diseases [59]. In contrast to other types of Cas13, Cas13d [60] and some examples of Cas13a do not require a Protospacer Flanking Sequence (PFS).…”