Abstract
Purpose :
Single nucleotide mutation can result in retinal degeneration and irreversible blindness in individuals, known as inherited retinal diseases (IRDs). Recent advancements in gene editing technologies, such as base editing, offer a promising avenue for addressing IRDs. While effective, the potential for unintended “off-target” of the genome has presented a substantial obstacle to clinical applications of gene editing therapy. Here, we sought to utilize a compact RNA base editor to correct disease-causing genetic mutation at the RNA level for treating IRDs.
Methods :
The catalytically inactive Cas13bt3 (dCas13bt3) was modified by truncating the C-terminal region to remove the catalytic HEPN2 domain and then fused with the deaminase domain of ADAR2 (ADAR2DD). A panel of guide RNAs (gRNA) with a length of 50nt was designed, placing the mismatch at positions of the on-target base, respectively, for targeting a nonsense Rpe65 mutation (Rpe65R44X, a mouse gene associated with human Leber Congenital Amaurosis). The editing efficiency was assessed by Sanger sequencing and the level of Rpe65 protein recovery was measured via Western blot analysis. The RNA sequencing was employed to identify transcriptome-wide RNA editing events in human retinal pigment epithelium cells (ARPE19).
Results :
The 50-24 and 50-26 gRNAs showed noticeable correction rates of up to 40% and 6%, respectively. No bystander effects were observed in the targeted region, indicating that the editing was specific to the on-target site. The recovery of the Rpe65 protein was detected following the editing event RNA editing events in ARPE19 cells. RNA-Seq analysis revealed that the expression of ADAR2DD led to an increase in the A-to-I editing events across the transcriptome, particularly on chromosome 19. Gene ontology analysis demonstrated that dCas13bt3-ADAR2DD with either a non-targeting or Rpe65R44X targeting gRNA enriches the α-interferon response, suggesting that the dCas13bt3-ADAR2DD-gRNA complex triggers cellular inflammatory pathways in ARPE19 cells.
Conclusions :
This study provides evidence for the precise and efficient correction of disease-causing mutations through the dCas13bt3-ADAR2DD RNA base editor. The frequent A-to-I editing events in the transcriptome indicate that the overexpression of the base editing effector may pose potential off-target effects. These findings contribute to the clinical development of RNA base editing for targeting IRDs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.