Journal: Molecular Therapy. Nucleic Acids
Article Title: Upregulation of a CFTR mRNA isoform has therapeutic potential for the treatment of 3′ CFTR PTC variants
doi: 10.1016/j.omtn.2025.102829
Figure Lengend Snippet: Therapeutic rationale for upregulation of a nonsense-mediated decay (NMD)-insensitive CFTR mRNA isoform for the treatment of 3′ CFTR PTC variants (A) Diagram of FL-WT, FL-W1282X, and e22 trunc mRNA isoforms. (B) Kinetics of the mRNA isoform abundances measured after treatment with actinomycin D; fitted exponential decay curves (dashed) with 95% confidence interval of the prediction (gray-filled bands), p values of the nonlinear least-squares fits and the fitted half-life times (t 1/2 ) with 95% confidence intervals are also shown. The NMD-driven decay of FL-W1282X transcript is too fast for a meaningful determination of its half-life on the timescale used in the experiment. (C) CFTR Cl − transport (conductance, G t ) time course measured in FRT cells overexpressing WT CFTR cDNA (vehicle only) or F508del, C832X, and e22 trunc cDNAs, in vehicle (−) or treated for 24 h with 3/3 μM ELX/TEZ (+); 1 μM IVA acutely added during TECC-24 assay, as indicated. FRT parental, 96 h post-transfection, treatment (apical and basolateral) 24 h prior to assay. (D) Summary transport activity metric extracted from time course data shown in (C): defined as ΔInhibitor (total change in conductivity [mS/cm 2 ] in response to addition of CFTR inhibitors, calculated relative to the plateau values reached after forskolin [Fsk] + IVA treatments). (E) Transepithelial electrical resistance, a widely accepted measurement reporting on the integrity of tight junctions in cell culture models of epithelial monolayers, was unaffected by WT, F508del, C832X, or e22 trunc transfections or treatment with 3/3 μM ELX/TEZ. (F) The rationale for exon 22/23 splice blocking to promote e22 trunc mRNA isoform as a therapeutic strategy for 3′ terminal CFTR PTCs downstream of exon 22. We hypothesize that inhibition of exon 22/23 splicing leads to retention of intron 22, including its ApA sites, thereby making them accessible for ApA usage. Utilization of intron 22 ApA sites generates a truncated mature CFTR mRNA lacking exon junction complexes downstream of the stop codon, allowing it to evade NMD. Consequently, this mechanism results in elevated levels of truncated CFTR transcripts. pA, polyadenylation site.
Article Snippet: Reverse transcription was completed using Oligo d(T)23 VN_T3 = 5′-GCAATTAACCCTCACTAAAGGTTTTTTTTTTTTTTTTTTTTTTTVN-3′ and ProtoScript II First Strand cDNA Synthesis Kit (New England Biolabs) according to manufacturer’s recommendations.
Techniques: Transfection, Activity Assay, Cell Culture, Blocking Assay, Inhibition
Genecopoeia is a verified supplier 
Genecopoeia manufactures this product 
![Therapeutic rationale for upregulation of a nonsense-mediated decay (NMD)-insensitive CFTR mRNA isoform for the treatment of 3′ CFTR PTC variants (A) Diagram of FL-WT, FL-W1282X, and e22 trunc mRNA isoforms. (B) Kinetics of the mRNA isoform abundances measured after treatment with actinomycin D; fitted exponential decay curves (dashed) with 95% confidence interval of the prediction (gray-filled bands), p values of the nonlinear least-squares fits and the fitted half-life times (t 1/2 ) with 95% confidence intervals are also shown. The NMD-driven decay of FL-W1282X transcript is too fast for a meaningful determination of its half-life on the timescale used in the experiment. (C) CFTR Cl − transport (conductance, G t ) time course measured in FRT cells overexpressing WT CFTR <t>cDNA</t> (vehicle only) or F508del, C832X, and e22 trunc cDNAs, in vehicle (−) or treated for 24 h with 3/3 μM ELX/TEZ (+); 1 μM IVA acutely added during TECC-24 assay, as indicated. FRT parental, 96 h post-transfection, treatment (apical and basolateral) 24 h prior to assay. (D) Summary transport activity metric extracted from time course data shown in (C): defined as ΔInhibitor (total change in conductivity [mS/cm 2 ] in response to addition of CFTR inhibitors, calculated relative to the plateau values reached after forskolin [Fsk] + IVA treatments). (E) Transepithelial electrical resistance, a widely accepted measurement reporting on the integrity of tight junctions in cell culture models of epithelial monolayers, was unaffected by WT, F508del, C832X, or e22 trunc transfections or treatment with 3/3 μM ELX/TEZ. (F) The rationale for exon 22/23 splice blocking to promote e22 trunc mRNA isoform as a therapeutic strategy for 3′ terminal CFTR PTCs downstream of exon 22. We hypothesize that inhibition of exon 22/23 splicing leads to retention of intron 22, including its ApA sites, thereby making them accessible for ApA usage. Utilization of intron 22 ApA sites generates a truncated mature CFTR mRNA lacking exon junction complexes downstream of the stop codon, allowing it to evade NMD. Consequently, this mechanism results in elevated levels of truncated CFTR transcripts. pA, polyadenylation site.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8342/pmc12858342/pmc12858342__gr2.jpg)
