Journal: Molecular Therapy Oncology

Article Title: Survivin/BIRC5-derived peptide disrupts survivin dimerization and cell division and induces multifaceted anti-cancer effects

doi: 10.1016/j.omton.2025.201123

Figure Lengend Snippet: Cell death induction by 1H13-survivin/BIRC5-derived peptides targeted to the cytoplasm, mitochondria, or nucleus (A) The sequences added to the 1H13-BIRC5 peptide to target it to the: (1) cytoplasm, (2) mitochondria, or (3) nucleus. The underlined sequence represents the 1H13 peptide; in red, amino acids indicate D-amino acid substitutions. (B) A549 cells were incubated for 90 min with 5 μM FITC-labeled, nucleus-targeted peptide IH13-Nuc, and were also IF-stained with anti--SMAC, anti-IP3R or anti-GM130 antibodies, and stained with DAPI to visualize the mitochondria, ER, Golgi, and nucleus, respectively. Confocal microscope images are shown, with white arrows indicating peptide co-localization with the mitochondria (SMAC). Orange and yellow arrows indicate peptide presence in the nucleus and cytosol, respectively. (C) A549 cells were incubated with the mitochondria- or nucleus-targeted 1H13-BIRC5-derived peptide for 24 h in serum-free medium, followed by a cell proliferation assay using the SRB method. (D and E) Apoptotic cell death as induced in A549 cells following incubation for 24 h with the nucleus-targeted peptide (2/3D-1H13-Nuc) in the presence or absence of the indicated concentrations of the peptides in serum-free medium and subjected to FITC–annexin V/PI staining, followed by a flow cytometry analysis. Representative histograms for control and selected peptide concentration (D) and analysis of early and late apoptotic stages are shown (E). (F and G) Cell death as induced by 2/3D-1H13-Nuc in different cell lines, A549, SH-SY5Y, U-87MG, PC-3, and HUV-EC-C (F) or Jurkat, K562, and KMH2-LC (G) were treated with the indicated concentrations of the peptide for 24 h, then subjected to cell death analysis using propidium iodide (PI) staining and flow cytometry. (H and I) A549 cells were seeded at a density of 2 × 10 5 cells per well in a 12-well plate. After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions. Twenty-four hours post-transfection, the cells were re-seeded at 1 × 10 5 cells per well in a 12-well plate. After another 24 h, the culture medium was replaced with serum-free medium, and the cells were treated with the indicated concentrations of the 2/3D-1H13-Nuc peptide. Survivin overexpression levels were assessed by immunoblotting (H), and cell death was analyzed by propidium iodide (PI) staining followed by FACS analysis (I). Results represent the means ± SEM ( n = 3).

Article Snippet: After 24 h, the cells were transfected with 2 μg of a pCMV3-survivin expression plasmid (HG10356-UT, Sino Biological, China) or with an empty pCMV3 plasmid (control) using JetPrime transfection reagent (Polyplus, France), following the manufacturer’s instructions.

Techniques: Derivative Assay, Sequencing, Incubation, Labeling, Staining, Microscopy, Proliferation Assay, Flow Cytometry, Control, Concentration Assay, Transfection, Expressing, Plasmid Preparation, Over Expression, Western Blot

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