Poster: 329Four models of the CFTR R553X mutation corrected by an adenine base editor delivered with HDAd

Y. Wang; K. Kulhankova; A. Loren; K. Thrasher; J. Lueck; C. Hemez; D. Liu; A. Cooney; P. McCray; P. Sinn

doi:10.1016/j.jcf.2026.07.359

Approximately 10% of individuals with CF carry nonsense, frameshift, or splice-site mutations that prevent them from benefiting from currently available CFTR modulator therapies. Adenine base editors (ABEs) have shown promise in correcting the CFTR R553X nonsense mutation; however, in vivo demonstration of efficacy has been limited by the lack of efficient delivery. The goal of this study was to evaluate the genetic and functional correction of the CFTR R553X mutation using a versatile helper-dependent adenoviral (HDAd)-based vector (HDAd-ABE8e-R553X). Here we demonstrate efficacy in 4 genetic models: (i) transduced reporter constructs, (ii) immortalized airway epithelial cells, (iii) primary airway epithelial cells from human CF lung donors, and (iv) primary mouse airway epithelial cells from a humanized CFTR R553X mouse model. 1) CFTR-R553X reporter construct. To evaluate the base-editing capacity of HDAd-ABE8e-R553X, we employed an EF1α-driven reporter construct (EF1α–NLS-mVenus-NanoLuc-mScarlet) that includes a human CFTR exon 11 fragment containing the R553X mutation inserted between the split NanoLuc luciferase subunits. Restoration of the full NanoLuc and mScarlet occurs only when the premature stop codon is corrected. Primary HAE cells were transduced with the lentiviral vector encoding the reporter system and subsequently transduced with HDAd-ABE8e-R553X. Finally, mScarlet and NanoLuc protein expression were quantified. 2) In vitro correction of CFTR R553X. 16HBEo- cells homozygous for the CFTR R553X mutation (16HBEge-R553X) were transduced with HDAd-ABE8e-R553X. High-throughput sequencing (HTS) was used to measure the correction of the R553X site. 3) Correction of primary human CF airway epithelia (HAE). Primary cells carrying the R553X mutation were differentiated at an air–liquid interface (ALI) to model physiological airway epithelium. HTS was utilized to determine the editing of R553X site. 4) In vivo correction of CFTR R553X. A humanized CFTR R553X mouse model from Cystic Fibrosis Mouse Models Core at Case Western Reserve University was generated by replacing mouse exon 11 with human exon 11 containing the R553X mutation. We isolated primary airway basal cells and differentiated the cells at ALI. The cells were transduced by HDAd-ABE8e-R553X. The functional and genetic correction was analyzed by Ussing chamber and HTS. [1] CFTR-R553X reporter construct. Robust expression of NanoLuc and mScarlet indicated successful editing in the HAE reporter system. [2] In vitro correction of CFTR R553X. Editing efficiencies in 16HBEge-R553X cells ranged from ∼20% to ∼90% in an MOI-dependent manner. [3] Correction of primary human CF airway epithelia (HAE). HDAd-ABE8e-R553X transduction resulted in 10–20% editing. These levels have been previously shown to restore CFTR function in ALI cultures, suggesting physiological relevance. [4] HDAd-ABE8e-R553X transduction corrected ∼20% CFTR R553X mutation which achieved ∼80% of CFTR functional correction as a chloride ion channel in primary mouse airway epithelial cells. These studies demonstrate that an HDAd vector delivering an adenine base editor can achieve robust correction of the CFTR R553X mutation across four complementary models. HDAd-ABE8e-R553X shows strong potential as a therapeutic platform for individuals with CF caused by nonsense CFTR mutations.

Poster: 329Four models of the CFTR R553X mutation corrected by an adenine base editor delivered with HDAd

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