Journal: bioRxiv

Article Title: Preclinical studies for plant-based oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice with transgenic tobacco seeds expressing human GAA (tobrhGAA)

doi: 10.1101/2021.11.11.468227

Figure Lengend Snippet: A human PD myoblast cell line was exposed to equivalent amounts of a lysate of tobrhGAA or an rhGAA for 48 hr and assayed for GAA and NAG. We found that tobrhGAA increased GAA to 24-35% of normal (mean±SD).

Article Snippet: Mock treated GAA and normal myoblast cells were controls plus cells treated with equivalent amounts of a rhGAA (R&D Systems #8329-GH-025).

Techniques:

Journal: Frontiers in Genetics

Article Title: Case Report: Incidental late-onset Pompe disease diagnosis in a man with no clinical and instrumental evidence of neuromuscular dysfunction

doi: 10.3389/fgene.2025.1574381

Figure Lengend Snippet: Genetic studies (A) Pedigree of the family investigated. Subjects harbouring IVS1-32-13T>G in homozygosis (+/+) or heterozygosis (+/−) are shown. The arrow indicates the Proband. (B) Reverse transcription PCR (RT-PCR) encompassing GAA transcript Exons 1 (E1) and 3 (E3) in lymphocytes of the subjects indicated. Amplicons corresponding to normally spliced molecules including Exon 2 (“E2” = Normal transcript “N”) and altered transcripts (Splice Variants “SV1-3”) are indicated. (C) Tape Station analysis of RT-PCR amplicons and their relative quantification. (D) Quantitative RT-PCR analysis of GAA transcripts in lymphocytes of the subjects indicated as evaluated by Taqman probes targeting Exons 1-2 and 4-5 junctions.

Article Snippet: The following probes were used: Hs00164635_m1 ( GAA , exon junction 1-2); Hs01089838_m1 ( GAA , exon junction 4-5); Hs99999903_m1 ( ACTB, encoding the housekeeping beta actin).

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: Case Report: Incidental late-onset Pompe disease diagnosis in a man with no clinical and instrumental evidence of neuromuscular dysfunction

doi: 10.3389/fgene.2025.1574381

Figure Lengend Snippet: Transcript and Protein analysis of Patients’ fibroblasts. (A) Reverse transcription PCR (RT-PCR) encompassing GAA transcript Exons 1 (E1) and 3 (E3) in Proband’s and control fibroblasts. Amplicons corresponding to normally spliced molecules including Exon 2, (“E2” = Normal transcript “N”) and altered transcripts (Splice Variants “SV1-3”) are indicated. (B) Tape Station analysis of RT-PCR amplicons and their relative quantification. (C) Quantitative RT-PCR analysis of GAA transcripts in Proband’s and control fibroblasts as evaluated by Taqman probes targeting Exons 1-2 and 4-5 junctions. (D) Western blot analysis of GAA protein levels in Proband’s and control fibroblasts. Mature forms of GAA enzyme correspond to bands at 76 and 70 kDa. GAPDH levels are used for normalization purpose.

Article Snippet: The following probes were used: Hs00164635_m1 ( GAA , exon junction 1-2); Hs01089838_m1 ( GAA , exon junction 4-5); Hs99999903_m1 ( ACTB, encoding the housekeeping beta actin).

Techniques: Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Control, Quantitative Proteomics, Quantitative RT-PCR, Western Blot

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: PPMO increases GAA enzyme activity in patient-derived fibroblasts (A) GAA mis-splicing caused by the common IVS1 mutation (red) compared with canonical GAA splicing (black). Splicing events were detected by endpoint RT-PCR. (B) Schematic depicting the location of PPMO compounds in microwalk screen. (C) Members of the microwalk screen were assayed for increases in GAA enzyme activity in LOPD patient fibroblasts. NTC = nontargeting control. One-way ANOVA with Dunnett’s multiple comparison test. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗<0.0001. (D) Optimization of hit compound sequence RC-3001 using an abasic subunit to address high GC content. (E) Abasic-containing sequence (RC-3003) restores GAA enzyme activity in patient fibroblasts. Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: Activity Assay, Derivative Assay, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Control, Comparison, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: LOPD patient iPSC-derived myotubes recapitulate splicing deficits observed in LOPD patients (A) LOPD patient fibroblasts were reprogrammed into iPSC lines and pluripotency was confirmed by immunostaining with OCT3/4. LOPD iPSCs were terminally differentiated into LOPD patient derived iPSC myoblasts and myotubes that express MYOD and MHC by immunostaining, respectively. PC, phase contrast. Scale bars: fibroblasts (500 μm), iPSC and myoblasts (100 μm), myotubes (200μm) (B) LOPD patient iPSC-derived myotubes faithfully recapitulate aberrant splicing reported in LOPD patients, including skipping of exon 2. C) GAA TV expression in LOPD patient iPSC-derived myotubes. TV1, NM_000152.5 ; TV2, NM_001079803.3 ; TV3, NM_001079804.3 . Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: Derivative Assay, Immunostaining, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: PPMO correct GAA splicing in LOPD patient iPSC-derived myotubes (A) PPMO treatment decreases pathogenic splicing and increases full-length GAA as measured by endpoint RT-PCR. Marker in basepairs. Full-length GAA , 1100 bp; exon 2 skipping, 450 bp, 550 bp. (B) PPMO compounds increase levels of GAA TV1 and TV2 upon treatment. (C) PPMO treatment precisely restores the LOPD-critical GAA exon 1–2 junction with no detectable undesired splicing variants as measured by RNA amplicon sequencing. Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: PPMO correct pathogenic GAA levels at transcript, protein, and lysosomal enzyme activity levels in LOPD patient iPSC-derived myotubes (A) PPMO treatment increases GAA TV1 and TV2 at the LOPD critical exon 1-2 junction and also increases. (B) Total GAA (all TVs) transcript levels as measured by qPCR. (C and D) PPMO treatment similarly increases GAA protein (one-way ANOVA with Dunnett’s multiple comparison test; ∗ p < 0.05; ∗∗ p < 0.01). Arrows indicate size of GAA primary translation (76 kDa) and post-translationally modified GAA (76 kDa). (E) Lysosomal GAA enzyme activity levels in patient iPSC-derived myotubes were increased after treatment with PPMO in a dose-dependent fashion. Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: Activity Assay, Derivative Assay, Comparison, Modification

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: Generation and characterization of a relevant murine model of LOPD (A) Human GAA harboring the IVS1 mutation was inserted into the Rosa26 safe harbor locus. (B) Single copy integration was confirmed by genomic qPCR. (C) GAA TV expression in skeletal muscle was confirmed by qPCR. (D) GAA expression was confirmed in various tissues by qPCR analysis. (E) Endpoint RT-PCR analysis demonstrated that the mutant human GAA transgene was faithfully mis-spliced in the mouse and produced the same GAA splice variants found in LOPD patients. (F) GAA mis-splicing in mouse quadricep muscle was confirmed by RNA amplicon sequencing. Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: Mutagenesis, Expressing, Reverse Transcription Polymerase Chain Reaction, Produced, Amplification, Sequencing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Splicing correction by peptide-conjugated morpholinos as a novel treatment for late-onset Pompe disease

doi: 10.1016/j.omtn.2025.102524

Figure Lengend Snippet: Systemic PPMO treatment corrects GAA splicing in vivo (A) Endpoint RT-PCR qualitatively showed an increase in full-length GAA after single IV dose of RC-3002. (B) Amplicon sequencing confirmed an increase in correctly spliced exon 1–2 junction upon treatment with RC-3002. (C) qPCR analysis of RNA extracted from quadricep muscle of animals treated with a single dose of RC-3003 demonstrated a dose-dependent increase in GAA transcript levels that was found to correlate with (D) compound tissue concentrations. (E) treatment was well tolerated. One-way ANOVA with Dunnett’s multiple comparison test. ∗∗ p < 0.01. Results are expressed as mean with error bars (SD).

Article Snippet: We amplified 1 μL of DNA in a PCR reaction containing an assay for human GAA detection on the VIC channel (Hs03961696_cn, Thermo Fisher Scientific) and mouse Tfrc (Thermo Fisher Scientific) on the VIC channel using Genotyping Master Mix (Thermo Fisher Scientific, Quantstudio 7 Pro).

Techniques: In Vivo, Reverse Transcription Polymerase Chain Reaction, Amplification, Sequencing, Comparison