Background: One in every 10 people with cystic fibrosis (PwCF) are still in need of a specific treatment. Genetic-based therapies are of special interest to this subset of PwCF, providing a potential one-time cure and avoiding a lifelong treatment. Several research groups including ours have reported efficient correction of many CFTR mutations by homology directed repair (HDR) and adenine base editing (ABE) [1, 2]. However, an important question remains to be answered – which cell types, and how many of them, need to be corrected to restore CFTR function to therapeutic levels? Patients carrying nonsense mutations are among those in need of a specific treatment. To date, 177 nonsense mutations have been identified in the CFTR gene, W1282X (c.3846G >A) being the second most common [3]. Nonsense mutations introduce an in-frame premature termination codon (PTC) into the CFTR gene, leading to mRNA degradation by nonsense mediated decay (NMD) and consequently very little or no full length functional CFTR protein is produced.
Methods: To address the question of “which cells and how many”, we developed a W1282X version of the BCi-NS1.1 cell line, an immortalized line that can differentiate into all known lung cell types. In parallel, we have developed a split SpRY-Cas9 Adenine Base Editor/gRNA (hereinafter split-ABE) with a ubiquitously expressed promoter (chicken β-actin promoter). This enables precision repair of W1282X (TGA to TGG) but only in the presence of rapamycin giving us temporal control of editing. Currently, we are developing a system for cell type-specific expression of the split-ABE initially under the expression of the KRT5 promoter for basal cell expression. In addition, we will use other cell type-specific promoters to restore W1282X in selected cells of the pulmonary epithelium. This approach will allow us to have spatial control of editing.
Results: The longer term goal is to grow these cells in air-liquid interface (ALI) culture, allow them to differentiate, and then edit in specific cell types. We will then seek to: i) determine the percentage of editing of all cell types in a mature pseudostratified epithelium (PE) that is necessary to restore CFTR function to WT levels; ii) correlate functional restoration of editing versus NMD inhibitors; iii) determine if editing basal cells alone is sufficient to restore CFTR function to WT levels; iv) determine if editing of other lung cell types is necessary/sufficient; v) determine if edited basal cells can repopulate a mature PE.
Conclusions: As a reporter system, our model should enable us to identify the pulmonary cell types in the CF disease pathogenesis that are necessary to target with a gene-editing approach to reverse the disease and may be of use to other research groups involved in the development and assessment of viral and non-viral delivery strategies. Importantly, our work may help accelerate the development of gene-editing therapies for CF towards clinics.
Acknowledgements: Work supported by grant HARRIS21G0 from CFF and centre grants UIDB/04046/2020 and UIDP/04046/2020 from FCT, Portugal (to BioISI).
References: [1] Santos L et al (2022) Comparison of Cas9 and Cas12a CRISPR editing methods to correct the W1282X-CFTR mutation. J Cyst Fibros. 21:181-187.
[2] Jiang T et al (2020) Chemical modifications of adenine base editor mRNA and guide RNA expand its application scope. Nat Commun. 11:1979. [3] CFTR2 (www.cftr2.org)
