(663) Modulation of CFTR intron 22 alternative polyadenylation (ApA) usage may have therapeutic potential for the treatment of certain 3’ CFTR PTC variants.

Background: Premature termination codon (PTC) mutations in the CFTR gene represent largest class of cystic fibrosis (CF) causing mutations for which there is no therapy addressing the underlying molecular mechanism. PTC mutations result in a poorly or non-functional protein product and also trigger a dramatic reduction of CFTR mRNA template via Nonsense Mediated Decay (NMD). C-terminally truncated CFTR protein, e.g. the product of the CFTR W1282X variant, retains some Cl- channel function. Therefore, inhibition of NMD to increase truncated protein expression would seem an attractive approach for PTC mutations near the 3’ end of CFTR. However, given the critical role of NMD as a global quality control mechanism of the cell, a more CFTR-specific mechanism to evade NMD is desirable. Here, we propose a novel approach to increase expression of a naturally occurring, lowly expressed alternatively polyadenylated (ApA) CFTR E22 3’ truncated mRNA that can evade NMD for CFTR variants carrying PTC mutations downstream of Exon 22, e.g., CFTR W1282X.

Methods: By gene-editing, models were created based on the 16HBE14o- cell line in which the splicing of CFTR exon 22/23 was inhibited or completely disrupted, promoting intron 22 ApA usage and resulting in increased expression of ApA CFTR E22 3’ truncated mRNA. Gene-edits were verified by full gene CFTR deep sequencing, RT corrected ddPCR was used to determine absolute ApA CFTR E22 3’ truncated mRNA copy number, protein expression was evaluated by Western blots and functional outcomes assessed with a TECC 24 assay with elexacaftor/tezacaftor/ivacaftor (Trikafta®).

Results: We created by gene-editing the intron 22 Splice Acceptor disrupted (SAd) 16HBEge CFTR W1282X cell line. As hypothesized, compared to the parental cell line, the deletion caused a 4-fold increase in the NMD-insensitive ApA CFTR E22 3’ truncated mRNA. A Exon 23 skipped mRNA and a W1282X FL-CFTR mRNA were also observed. The RNA stability determined from an Actinomycin D time course was much greater for the ApA CFTR E22 3’ truncated mRNA t_1/2 (4.24 h) compared to the W1282X FL-CFTR mRNA t_1/2 (< < < 2 h). Both Exon 23 skipped and WT FL-CFTR mRNA had a t_1/2 ~10.5 h. Interestingly, we observed near equal steady state levels for both Exon 23 skipped and ApA CFTR E22 3’ truncated mRNA irrespective of their dramatically different t_1/2, suggesting that ApA CFTR E22 3’ truncated mRNA may be processed from nascent to mature mRNA faster than Exon 23 skipped or WT CFTR mRNA.

To test if the CFTR E22 C-terminal truncated protein was responsive to modulators, it was heterologously expressed in FRT cells. Trikafta treatment yielded Cl^- current of ~15% of WT CFTR transfected cells.

To assess the maximum therapeutic potential of ApA CFTR E22 3’ truncated mRNA expression, a gene edited cell line was created with an Exon 23-3’UTR partial CFTR gene deletion that forced production of ApA E22 3’ truncated mRNA. Three of the resulting clones contained the correct ~27KB deletion of Exons 23-3’UTR on the functional allele, and truncated Band B and Band C of expected sizes were detected by Western blotting. The ApA CFTR E22 3’ truncated mRNA was 12-fold increased compared to parental, and resulted in 16.5-24.6% CFTR WT function in a TECC-24 assay.

Conclusions: Our data suggest inhibition of E22/23 splicing to promote expression of a CFTR E22 3’ truncated mRNA that escapes NMD may have therapeutic potential for PTC mutations downstream of Exon 22.

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