(603) Base editing rescues G542X CFTR mutation

Background: G542X is the second most common CF-causing mutation, with over 3,600 alleles in the CFTR2 database (www.cftr2.org). The single nucleotide change in CFTR generates a premature termination codon (PTC) which also results in nonsense-mediated decay (NMD), the overall result being almost no CFTR protein expression. As a consequence, the current modulators are ineffective against G542X. Our group has previously shown efficient gene and base editing of another CFTR PTC variant, W1282X, resulting in restoration of CFTR mRNA and protein, and partial rescue of CFTR function [1]. Here, we describe a base editing approach to correct G542X.

Methods: CRISPR-Base editing of G542X was first tested in a plasmid-based system using a target plasmid containing the CFTR cDNA with c.1624G >T (G542X) mutation. The target plasmid, ABE plasmid and specific guideRNA (gRNA) plasmid were transfected into HEK293 cells using Lipofectamine. Ninety-six hours later, DNA was extracted from cells and the target region amplified by PCR and Sanger sequenced. Editing efficiency was measured using the EDIT-R software (https://moriaritylab.shinyapps.io/editr_v10/). Three cell lines are also available with this mutation, and ABE here requires transfection only with the ABE and specific gRNA plasmids. To measure CFTR correction, quantitative real-time PCR, Western Blot and functional assays will be used to evaluate CFTR transcript and protein function restoration as previously described in our lab [1].

Results: At the DNA level, the G542X mutation is c.1624G >T, so our design strategy is to target the A on the non-coding strand and use adenine base editing (ABE) to convert this to G, the overall result being conversion of the TGA (stop) codon to CGA (Arg). Although this correction does not restore the WT amino acid, it could still recover partial CFTR function. Previous studies [2] have, indeed, shown through PTC suppression that correction to G542R restores full-length CFTR maturation to ~30% the wild-type (WT) protein and channel activity up to 40%. We opted for ABE8e-NG to induce the desired A >G transition, as this base editor has been shown to be highly efficient, especially in our editing window of interest. Base editing of G542X in a plasmid-based system, with ABE8e-NG and our custom-designed gRNA, yielded up to 30% ABE, resulting in correction of G542X to G542R in successfully transfected cells. In contrast to our studies with W1282X base editing, no bystander off-target bases were detected, which means the surrounding coding sequence is not negatively impacted.

Conclusions: The current focus is now to optimize G542X base editing in FRT, 16HBE14o-, HEK-CFTR Flp-In models, and primary cell lines, measure the level of CFTR functional rescue and determine any evidence of bystander edits. The potential for synergistic effects of modulators will be tested on edited cells with G542R, and delivery strategies evaluated, to establish a new treatment option for people with G542X.

Acknowledgements:

References: [1] Santos L, Mention K, Cavusoglu-Doran K, Sanz DJ, Bacalhau M, Lopes-Pacheco M, et al. Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation. J Cyst Fibros. 2022 Jan;21(1):181-187. doi: 10.1016/j.jcf.2021.05.014

[2] Roy B, Friesen WJ, Tomizawa Y, Leszyk JD, Zhuo J, Johnson B, et al. Ataluren stimulates ribosomal selection of near-cognate tRNAs to promote nonsense suppression. Proc Natl Acad Sci U S A. 2016 Nov 1;113(44):12508-12513. doi: 10.1073/pnas.1605336113