WS17.02 Improved adenine base editing approach to correct W1282X-CFTR

Aim: W1282X is the sixth most common cystic fibrosis (CF)-causing variant and one of those without an approved therapy. Adenine base editing (ABE) has attracted interest as it can correct mutations creating a double-stranded break in DNA, being thus potentially safer than traditional approach with CRISPR/Cas9. Efficient correction by ABE been reported but bystander edits have observed at adjacent adenines. Methods: To minimize duration nuclease activity reduce effects, we designed two new fusion proteins - Cas9(C)-NLS Cas9(N)-FRB developed novel (hereinafter split-ABE), which only dimerise function presence rapamycin. test split-ABE, generated version BCi-NS1.1 cells, cell line that differentiate into all known lung types. After electroporation split-ABE or SpRY-ABE (non-split control) plasmids, cells were treated rapamycin DMSO for 72 h. Editing efficiency was measured next-generation sequencing. Results show 4-fold increase levels compared to DMSO. Comparison non-split editors showed higher total A > G (14.7%), while 2.1% editing. Despite lower unintended bystanders almost nonexistent (≤ 0.5%) where close 7%. Conclusion: We improved ABE, corrects W1282X-CFTR substantially reduces edits. The fact be temporarily regulated small molecule drug gives us temporal control This will used improve gene therapeutic approaches terms safety target identification. Acknowledgements: Work supported grant HARRIS21G0 from CFF centre grants UIDB/04046/2020 UIDP/04046/2020 FCT, Portugal (to BioISI).