protoscript ii first strand cdna synthesis kit  (New England Biolabs)

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

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: Inhibition of exon 22 splicing forces intron 22 ApA usage and elevates e22 trunc mRNA and protein levels that can be corrected with CFTR modulators to therapeutically relevant levels (A) Edited CFTR gene (ΔE23-3′ UTR) to “force” e22 trunc mRNA expression in 16HBE14o- parental cells. (B) ddPCR absolute mRNA copies of FL-WT exon 25/26 (black) and e22 trunc (gray) from 16HBE14o- parental cells and 16HBEge-ΔE23-3′ UTR- clonal lines 2-H07, 3-B09, and 3-D01. (C) Western blot analysis of 16HBE14o- parental line ± ELX/TEZ and clones 2-H07, 3-B09, and 3-D01 ± ELX/TEZ. Antibody UNC 596 detects CFTR; ACTB serves as a loading control. (D) Western blot of PNGaseF-treated (de-glycosylated) FL-WT, FL-W1282X, and e22 trunc cDNA overexpression from HEK293 cells and 16HBEgeΔE23-3′ UTR-2-H07 and 16HBE14o- parental cells. Samples were diluted to produce equivalent band intensity. (E) Electrophysiological traces of 16HBEgeΔE23-3′ UTR gene-edited cell line clones 2-H07, 3-B09, and 3-D01 from TECC-24 assay after DMSO (vehicle) treatment (three upper panels) or 48 h treatment with ELX/TEZ (3/3 μM) (three bottom panels). All samples treated in-assay with IVA (1 μM). (F) TECC-24 I eq assay results of 16HBE14o- (gray bar) and 16HBEge W1282X (light gray bars) and IVA dose escalation in 16HBEgeΔE23-3′ UTR -2-H07 (black bars) ± ELX/TEZ 3/3 μM. AUC FSK+IVA is area under the curve calculated starting at the IVA addition time point in electrophysiological traces as shown in (E).

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: Inhibition, Expressing, Western Blot, Clone Assay, Control, Over Expression