Abstract
Purpose :
A degron refers to a protein fragment capable of targeting proteins to the ubiquitin-proteasome degradation pathway. The dTag13 ligand is a heterobifunctional inducer of FKB12F36V-CRBN dimerization. A dTag13 inducible degron was tethered to the N terminus of a prime editor split across dual AAV vectors. The ability to degrade CRISPR protein after sufficient editing has occurred could limit the accumulation of off-target edits.
Methods :
1) HEK293T cells were seeded and transfected with plasmids encoding pegRNA and degron tagged prime editor alongside a luciferase plasmid containing a targetable G>A nonsense mutation. Incremental doses of dTag13 ligand were added to the culture media at 1 hour or 72 hours following transfection. Prime editing of the G>A mutation was investigated with a dual luciferase assay at 96 hours. 2) 1000nM of dTag13 ligand with a biotin tag was injected intravitreally into a wild-type mouse. Eyes were harvested at sequential time points following injection and processed for IHC. 3) Three weeks following subretinal injection of degron tagged prime editor, dTag13 ligand was injected intravitreally into wild-type mouse eyes. Two days following intravitreal injection, eyes were harvested, and retinal lysates processed by western blot. 4) A TUNEL assay alongside OCT imaging were performed following intravitreal injection to assess ligand-mediated toxicity.
Results :
Significant editing (15.7%±0.84) of the luciferase plasmid was observed following addition of ≥ 25nM ligand at 72 hours but abolished after addition of ligand at 1 hour post transfection. This indicates that successful degradation of prime editor at early time points impaired editing. IHC confirmed the presence of dTag13 ligand within the retinal layers 30 minutes following intravitreal injection. Western blot performed on retinal lysates demonstrated absent SpCas9 immunoreactivity following injection of dTag13 at doses ≥1000nM. A faint protein band was detectable at the 500nM dose. There was no significant increase in TUNEL positive nuclei and no significant reduction in retinal thickness following injection of 10µM ligand compared to PBS injected controls at 2-weeks following injection.
Conclusions :
The efficacy and safety of the ligand dTag13 to mediate degradation of a degron tagged prime editor was confirmed in vivo. This finding enhances the safety profile of prime editing technology applied in a therapeutic context.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.