Antisense therapy targeting valosin-containing protein improves muscle damage and molecular problems in cell and mouse models of multisystem protein disease

We developed a cellular myopathy model of VCP disease by differentiating hiPSC carrying the VCP p.R155H variants to SMPCs and assessed the efficacy of VCP ASOs in these cells. Figure 1A illustrates the timeline and major milestones of hiPSC differentiation and maturation into myotubes. It has been previously reported that directed differentiation of any hiPSC line can be achieved through line‐specific tailoring of initial seeding density and addition of the small molecule CHIR99021, an agonist of the Wnt signalling pathway. Indeed, both cell seeding density and Wnt activation play a vital role in mesoderm formation.35, 36 For VCP hiPSCs, we found that 60 000 cells/cm² and supplementation of 8 µM CHIR99021 for 2 days sufficiently induced mesoderm and later myogenic differentiation, as noted by the formation of distinct 3D structures 1 week after mesoderm induction, and later juxtaposed myotube formation (Figure 1B). At the completion of directed differentiation, enrichment of SMPCs was achieved by selection of the cell population with the highest expression of the cell surface receptors erb‐b2 receptor tyrosine kinase 3 (ERBB3) and nerve growth factor receptor (NGFR) using fluorescence‐activated cell sorting (FACS)37 (Figure 1C). We found that co‐expression of ERBB3 and NGFR could sufficiently enrich SMPCs from VCP R155H cells, and these SMPCs robustly expressed Paired box protein 7 (PAX7) (Figures 1D and S1A). SMPCs could be differentiated to form myosin‐positive multinucleated myotubes, suggesting successful enrichment (Figure 1D).37 However, VCP R155H SMPCs displayed reduced myotube fusion efficiency compared with the control SMPCs (p < .05) (Figure 1E), which may be indicative of their diseased state which we quantified in subsequent figures.

Pathogenic VCP R155H variants are known to lead to the accumulation of ubiquitinated proteins, disruption of autophagy and impaired retrotranslocation of endoplasmic reticulum‐associated degradation substrates.38, 39 Therefore, we sought to determine the pathophysiological effects of VCP R155H pathogenic variants in the patient‐derived SMPCs. It has been reported that dysregulation of protein homeostasis due to variants in VCP may cause TDP‐43 to undergo cleavage, hyperphosphorylation and ubiquitination, leading to its accumulation and aggregation in the cytoplasm.15, 40, 41, 42 Phosphorylated TDP‐43 (p‐TDP‐43) is particularly associated with the pathological inclusions seen in TDP‐43 proteinopathies. We found TDP‐43 and VCP gene expression increased significantly by 1.9 ± 0.5‐fold and 1.9 ± 0.3‐fold, respectively, in VCP R155H cells compared with wild type (WT) animals (Figure 2A). The expression of the autophagic proteins sequestome‐1 (SQSTM1/p62) and light chain 3B (LC3B) in VCP R155H cells were 0.3 ± 0.08‐fold and 0.6 ± 0.07‐fold lower, respectively, compared with WT cells, as measured by qRT‐PCR analysis (Figure 2A). In addition, TDP‐43 and p‐TDP‐43 protein levels were significantly increased by 1.5 ± 0.29‐fold and 2.1 ± 1.32‐fold in VCP R155H cells compared with WT cells (Figure 2B, C).

LC3B is a key protein of the autophagy pathway that can be present in two different forms: a cytosolic form (LC3B‐I) and a lipidated form (known as LC3B‐II), where LC3B‐I is conjugated to phosphatidylethanolamine to form LC3B‐phosphatidylethanolamine conjugate, which is bound to the autophagosome membrane.43 The ratio of LC3B‐II/I reflect the conversion of LC3B‐I to LC3B‐II upon autophagy activation. We noted increased expression of LC3B‐I by 2 ± 0.73‐fold and LC3B‐II by 3.7 ± 2‐fold, respectively in VCP R155H cells compared with WT (Figure 2B, D). The LC3 II/I ratio clearly showed enhanced autophagic flux in VCP R155H cells. A 1.7 ± 0.5‐fold elevation in the levels of the autophagic marker SQSTM1/p62 also suggested impaired autophagosome‐lysosomal cascade in the VCP R155H cells (Figure 2B, D). Increased VCP protein expression by 1.6 ± 0.39‐fold was also observed in VCP R155H cells (Figure 2B, C). Through nuclear‐cytoplasmic fractionation, we observed significantly elevated cytoplasmic while reduced nuclear TDP‐43 levels by 1.37 ± 0.13‐fold and 1 ± 1.15‐fold, respectively, in VCP R155H SMPCs, implying possible TDP‐43 mislocalisation (Figure 2E–G). In agreement with the Western blot analysis, immunofluorescence data showed significant increase in TDP‐43 (Figure 2H, I) and p‐TDP‐43 (Figure 2J). The VCP R155H cells also showed increased SQSTM1/p62, LC3B and ubiquitin positive expression in the immunofluorescence analysis, therefore consolidating the protein expression data (Figure 2J). The VCP R155H SMPCs thus manifest a phenotype characterised by arrested autophagy and increased TDP‐43 expression, providing a useful model to study the effect of ASO treatment in MSP1 disease.

VCP plays a crucial role in maintaining lysosomal homeostasis.44, 45 The disruption of lysophagy due to VCP pathogenic variants leads to lysosomal accumulation, suggesting a defective lysosomal membrane permeabilisation (LMP) response in the development of VCP‐associated MSP1. VCP R155H and WT cells were subjected to starvation and a combinatorial treatment with bafilomycin A1 (BafA1), an inhibitor of autophagosome‐lysosome fusion, to assess autophagic flux and accumulation of autophagosomes.46, 47 In VCP R155H cells, impaired autophagy led to a significant increase in SQSTM1/p62 protein levels upon BafA1 treatment (Figure 3A, B). LC3B‐I levels were also significantly higher in BafA1‐treated VCP R155H cells compared with WT (Figure 3A, C). During autophagic induction, LC3B‐II is incorporated into the growing autophagosome membrane, and its level is proportional to the number of autophagosomes in the cell. Western blot analysis revealed that LC3B‐II level in WT did not change upon starvation, but was significantly enhanced after BafA1 treatment, indicating activated autophagic flux (Figure 3A, D). VCP R155H cells also showed a significant increase in LC3B‐II levels upon BafA1 treatment (Figure 3A, D). However, treatment with BafA1 resulted in significantly higher level of LC3‐II and the ratio of LC3B‐II/I in VCP R155H cells compared with WT (Figure 3E). After 24 h of recovery, the LC3B‐II/I ratio returned to basal levels in WT cells (Figure 3E). On the other hand, a higher LC3B‐II/I ratio persisted in VCP R155H cells due to disrupted autophagy (Figure 3E). In line with the Western blot data, immunofluorescence staining showed that the number of SQSTM1/p62‐positive dots was higher in starved VCP R155H cells and significantly increased in the presence of BafA1 (Figure 3F, G). Similarly, starvation led to a marked increase in the number of LC3B‐positive puncta, which was further amplified with BafA1 treatment in VCP R155H cells (Figure 3H).

Lysosomal damage in VCP R155H cells was further examined by analysing galectin‐3 (Gal‐3), a β‐galactoside‐binding lectin that is recruited to the lysosomal membrane solely upon membrane permeabilisation. In WT cells under basal conditions, Gal‐3 exhibited a diffuse cytoplasmic distribution with no detectable puncta (Figure 3I); however, starvation‐induced lysosomal damage resulted in the translocation of Gal‐3, and its consequent binding to compromised lysosomes, forming distinct puncta on the damaged lysosomal membrane. We observed that VCP R155H cells expressed significantly higher Gal‐3 puncta in the basal state (Figure 3I, J). Upon starvation, the number of Gal‐3 puncta increased in both WT and R155H cells due to lysosomal damage. Upon BafA1 treatment the number of Gal‐3 puncta in VCP R155H cells was significantly higher, compared with WT. These results show that VCP R155H patient cells have alterations in lysosomal stability and dynamics.

To investigate the therapeutic potential of VCP ASO in rescuing disease phenotypes in VCP R155H cells, we first examined the cytotoxicity of ASOs on the VCP R155H SMPCs at doses ranging from 50 nM to 5 µM. A cell survival rate of ≥98 % was measured in cytotoxicity studies where VCP R155H SMPCs had been treated with ASO up to 5 µM (Figure 4A). No significant differences were observed between control ASO and VCP ASO treatment. The VCP ASO was designed to specifically knock down VCP mRNA using RNase H1‐mediated degradation. Indeed, treatment with 1.2 µM VCP ASO reduced VCP protein expression by 50% (95% CI [19–68]) in the cells, whereas control ASO demonstrated no change in VCP protein levels (Figure 4B). To evaluate potential off target effects, VCP R155H SMPCs were treated with varying concentrations of control ASO. Western blot analysis revealed that control ASO treatment did not affect VCP protein levels (Figure S2A, B). Furthermore, TDP‐43, p62 and LC3B protein expression remained unchanged across all control ASO concentrations, indicating minimal off‐target effects (Figure S2A,C–G). Based on this promising result, we sought to further explore the relationship between different levels of VCP reduction and improvements in disease traits by conducting a dose–response experiment. The SMPCs were treated with four different concentrations of ASOs: 0.3, 0.6, 0.9 and 1.2 µM, to correlate the level of ASO‐mediated VCP mRNA and protein reduction with various molecular signatures that are characteristic of MSP1 disease pathology. We initially treated SMPCs with VCP ASO for 3 days, and Western blot analysis showed no significant changes in VCP, TDP‐43, p62 or LC3B protein levels (Figure S2H, J–N). We hypothesise that the 3‐day treatment period was insufficient to affect these downstream markers, which are characteristic of VCP‐associated pathology. To further investigate, we extended the ASO treatment duration to 7 days. qRT‐PCR analysis and Western blot analysis of 7‐day treatments revealed that ASO treatment resulted in 85% (95% CI [58–93]) reduction in VCP mRNA level and 48% (95% CI [39–56]) reduction in VCP protein expression, respectively, with no significant changes between the different ASO concentrations (Figure 4C, E,F). We observed increased LC3B mRNA and ratio of LC3B‐II/I protein expression upon ASO treatment, which may suggest activation of autophagic flux (Figure 4D, K). Furthermore, VCP ASO treatment resulted in a declining trend (not significant, except for 1.2 µM) of SQSTM1/p62 protein (Figure 4H), despite an increase in its mRNA levels (Figure 4D), which agrees with the notion that SQSTM1/p62 is degraded via autophagy. TDP‐43 protein expression decreased by 60% (95% CI [34–58]) upon ASO treatment compared with untreated cells (Figure 4G). Immunofluorescence confirmed the ASO‐mediated knockdown of VCP expression in VCP R155H cells and associated reduction in pTDP‐43 levels (Figure 4L). Consistent with the Western blot data, the immunofluorescence analysis also showed that VCP ASO treatment resulted in up‐regulation of LC3 and modest down‐regulation of SQSTM1/p62 protein levels, indicating initiation of autophagy (Figure 4M). Total ATPase measurements in VCPR155H SMPCs demonstrated a modest trending toward WT levels following VCP ASO treatment.

Studies have shown that VCP inhibition in mutant VCP cells rescues defective lysophagy.48 We utilised VCP ASO to accelerate clearance and prevent the persistence of damaged lysosomes in VCP R155H cells. We observed a declining trend in SQSTM1/p62 protein expression upon ASO treatment (Figure 4H). To investigate whether p62 degradation was mediated by autophagy, VCP R155H cells were co‐treated with 0.6 µM VCP ASO and Baf A1 (Figure 5A, B). This combination inhibited p62 clearance, which was initially observed with 0.6 µM VCP ASO alone (Figure 4H). Furthermore, p62 expression returned to baseline levels 24 h after Baf A1 washout in VCP R155H cells treated with VCP ASO, implicating autophagic contribution to p62 degradation. Additionally, we noted that the increased expression of LC3B‐II following 0.6 µM VCP ASO treatment was further elevated when autophagosome degradation was blocked by Baf A1 treatment (Figure 5A, C). Immunofluorescence staining also showed BafA1 treatment increased the number of SQSTM/p62 dots in VCP R155H cells treated with 0.6 µM VCP ASO, suggesting blockage in p62 degradation (Figure 5D, E). At basal level, in VCP R155H cells treated with 0.6 µM VCP ASO, Gal‐3 showed uniform cytoplasmic localisation without detectable puncta, signifying the possible removal of damaged lysosomes through autophagy (Figure 5F, G)). VCP R155H cells without ASO treatment had higher Gal‐3 puncta (Figure 5F, G)). Upon starvation and BafA1 treatment, the number of Gal‐3 puncta increased in both treated and untreated VCP R155H cells (Figure 5F, G). VCP being a key regulator of lysophagy, its targeted inhibition by VCP ASO in VCP R155H cells can potentially rescue lysophagic defects.

We conducted in vivo studies using a transgenic humanised mouse model of VCP disease harbouring the most severe mutation, VCP A232E (gifted by Paul Taylor).49 To initially generate transgenic mice expressing normal human VCP in all tissues, human VCP cDNA was placed under the control of the CMV‐enhanced chicken beta‐actin promoter permitting widespread VCP expression in muscle, brain and bone. The disease‐associated mutation A232E was then introduced by site‐directed mutagenesis. The resulting transgenic animals were backcrossed onto the C57BL/6 background. The translational value of the VCP A232E transgenic mouse model has been characterised in previous studies.49 The mice exhibited progressive muscle weakness; histological analyses of the quadricep muscle revealed signs of myogenic myopathy, such as irregular fibre size, centrally located nuclei and inflammatory cell infiltration; and histopathological analysis of the brain demonstrated ubiquitin‐positive cytoplasmic accumulations of TDP‐43. We additionally performed a natural history study in these mice in our laboratory and found differences between WT and VCP A232E mice in functional studies, including Rotarod, grip strength and inverted screen. Biochemical analysis of quadriceps, tibialis anterior and diaphragm muscle tissue revealed TDP‐43 pathogenesis and impaired autophagy similar to the previous report.49

To ensure the absence of ASO‐related toxicity in the mouse studies, 10‐month‐old VCP A232E mice were dosed with either control or VCP ASOs at 50 mg/kg, once a week for 12 weeks, via subcutaneous injection. Age and sex‐matched WT mice were used as a reference control (Figure). Percent change of body weights, illustrating relative changes in body weight over time, was calculated from a 6‐month‐old baseline (pre‐treatment) and set to zero for visualisation. At the end of the 12‐week dosing regimen, liver, spleen and kidneys were collected from the mice, and their weights were measured and analysed for potential toxicity. The serum levels of the liver transaminases aspartate aminotransferase (AST) and alanine aminotransferase (ALT), creatine kinase (CK), as well as the levels of blood urea nitrogen (BUN) whose elevation (more than twofold) would indicate concerning liver, muscle or renal toxicity, were also measured (Figure). We observed no significant changes in organ weights or serum parameters between VCP ASO and control ASO treatment groups at 12 weeks following injections using Dunnett's multiple comparisons test, except for BUN levels; however, all levels remained in the normal range (Figure). Therefore, we conclude that under the experimental conditions used, the VCP ASO was well tolerated in mice. S3A, B S3B–H S3B–H

Motor strength and coordination were assessed monthly in VCP A232E mice that received subcutaneous injections of 50 mg/kg VCP ASO or control ASO once a week from 6 to 9 months of age (Figure 6A). The 6‐month timepoint represents baseline motor performance prior to ASO treatment. A group of age‐matched untreated WT mice was included in the study as reference controls. Motor performance in the different groups of mice was evaluated with the Rotarod, grip strength and inverted screen assays, and both raw data and percent change from the respective baseline values were analysed. VCP A232E mutant mice dosed with VCP ASO showed significant improvement in Rotarod performance compared with control ASO‐treated A232E mice at 2 months of treatment (Figure 6B). Motor testing data trended for improved performance in the inverted screen test with VCP ASO treatment in A232E mice but did not reach statistical significance (Figure 6D). Percent change analysis of rotarod and inverted screen tests showed significant improvement in VCP ASO‐treated mice after 2 and 3 months of treatment when compared with the control ASO‐treated mice (Figure 6C, E). While the absolute differences between time points are modest (Figure 6B, D), the percent change graph highlights relative trends in motor performance over time. There was no change in grip strength performance (Figure 6F, G). To provide an integrated view of the cumulative effect of ASO treatment across the intervention period, we conducted statistical analysis using area under the curve (AUC) tests for the rotarod, inverted screen, grip strength and their respective percent change analyses. We compared the three groups AUCs using one‐way ANOVA and found a significant difference in inverted screen percent change AUC between VCP A232E treated with VCP ASO and control ASO (p < .0001). We did not find significant differences of AUCs between VCP ASO and control ASO‐treated groups for rotarod, inverted screen, grip strength, rotarod percent change or grip strength percent change (Figure S4A–F).

Taylor et al.49 reported that VCP A232E mice muscle exhibit pathological signs of myogenic myopathy, such as fibre size irregularity, centralised nuclei, inflammation and TDP‐43‐positive intranuclear and cytoplasmic aggregates. Consistent with those reports, we observed that 9‐month‐old VCP A232E mice treated with control ASO had significantly higher TDP‐43 mRNA (2.69 ± 1.4‐fold elevation) and protein (1.3 ± 0.07‐fold increase) expression in quadriceps muscle compared with WT littermates. Treatment with VCP ASO significantly reduced TDP‐43 mRNA levels by 0.47 ± 0.08‐fold and protein expression by 0.75 ± 0.03‐fold compared with control ASO in quadriceps muscle of VCP A232E mice (Figure 7A, B, D). SQSTM1/p62 and LC3B mRNA expression were 4 ± 0.98‐fold and 6.6 ± 0.2‐fold higher, respectively in quadriceps muscle of VCP A232E mice treated with control ASO compared with WT (Figure 7A). Treatment with VCP ASOs decreased SQSTM1/p62 and LC3B mRNA expression by 1.3 ± 0.43‐fold and 0.6 ± 0.3‐fold, respectively compared with control ASO treatment (Figure 7A). Similarly, SQSTM1/p62 and LC3B protein levels were reduced by 22% (95% CI [13–31]) and 45% (95% CI [32–55]) following VCP ASO dosing in quadriceps muscle of VCP A232E mice compared with control ASO (Figure 7B, E–H). The conversion of the soluble LC3B‐I protein to its lipidated, membrane‐bound LC3B‐II form represents a critical step in autophagosome formation. Therefore, the absence of LC3B‐II in control ASO‐treated VCP‐A232E mice indicates an impairment in the early stages of autophagy (Figure 7H). Additionally, VCP ASO dosed for 12 weeks at 50 mg/kg via subcutaneous injection resulted in 0.88 ± 0.1‐fold knockdown of VCP mRNA and a 30% (95% CI [27–32]) reduction in VCP protein expression compared with control ASO in quadriceps muscle of VCP A232E mice (Figure 7A–C). Haematoxylin and eosin (H&E) staining of histological sections of quadriceps muscle from VCP A232E mice dosed with VCP ASO showed improved myofibre morphology compared with control ASO (Figure 7I). Indeed, WT mice had homogeneously sized myofibres (Feret's diameter and cross‐sectional area: 57 ± 3.28 µm and 3289 ± 323 µm²) with peripherally located nuclei (Figure 7J,–L), whereas 9‐month‐old VCP A232E mice dosed with control ASO showed cellular infiltrations into the interstitium of the myofibres along with a significantly increased number of centralised nuclei, consistent with MSP1 myopathy (Feret's diameter and cross‐sectional area: 32 ± 2.23 µm and 1163 ± 143 µm²) (Figure 7K, L). VCP ASO treatment significantly decreased the number of central nuclei compared with control ASO (WT: 12 ± 2% myofibres had centralised nuclei, control ASO: 34 ± 2% myofibres had centralised nuclei, VCP ASO: 20 ± 2% myofibres had centralised nuclei; over 150 myofibres examined from 3 mice per group) (Figure 7J). VCP ASO treatment also significantly increased Feret's diameter and cross‐sectional area by 46 ± 2.56 µm and 2295 ± 250 µm² respectively, indicating potential therapeutic benefit.

We differentiated iPSCs of one clinically affected female patient (aged 65 years) carrying the VCP p.R155H variants to SMPCs using a previously published protocol.35 The patient developed myopathy with onset at the age of 33 years; Paget disease with onset at 37 years of age. Briefly, iPSCs were cultured on mTESR⁺ for at least three passages. After pre‐treating the iPSCs for 1 h with 10 µM Rho associated kinase inhibitor (rock inhibitor; RI) (Stem cell Technologies) supplemented media, cells were dissociated, seeded at a concentration of 550 000 cells/well (based on our standardisation protocol) on Matrigel‐coated plates and grown in mTeSR⁺ with RI media. On days 2 and 3, cells were treated with 8 µM CHIR (Tocris Biosciences) supplemented in E6 medium. Afterwards, the culture medium was changed, and cells were cultured in E6 medium until day 11. Cells were then switched to StemPro‐34⁺basic fibroblast growth factor media for an additional 7 days. The culture medium was then changed to E6 medium supplemented with IGF1 for 7 days. The cells were then fed Dulbecco's modified Eagle medium (DMEM/F12) with N‐2 Supplement (Gibco), insulin–transferrin–selenium (ITS; Gibco) and IGF1 media for 5–7 days and thereafter SB431542 (Sigma) was added for an additional 7 days. FACS was then conducted using mouse anti‐human fluorochrome‐conjugated monoclonal antibodies of erbB3/HER‐3 (Biolegend; 324705) and NGFR (CD 271; BD Pharmingen™ 562123). Briefly, the cells were dissociated as single cells, incubated in human Fc block antibody to block non‐specific binding of Fc, and subsequently fluorochrome‐conjugated antibodies were added according to previously published protocol.35 After 45 min incubation, the cell–antibody conjugates were washed with FACS buffer (2% fetal bovine serum in sterile phosphate‐buffered saline [PBS]) and analysed with BD ARIA II (BD Biosciences) flow cytometer and analysed with the BD FACS Diva 8.0.2 software. Matched fluorophare‐conjugated isotype control antibodies were used to set control gates. The sorted SMPCs were expanded in SMPC expansion media SkGM‐2 Bullet Kit (Lonza) for characterisation and further experimentation. The SMPCs were differentiated to myotubes by seeding them on Matrigel‐coated plates and culturing them in DMEM/F12 medium supplemented with N‐2 Supplement, ITS, IGF1 and SB431542 for 5–7 days. To evaluate the differentiation and myotube formation ability of SMPC, cultures were immunostained with MF 20 and PAX7 antibodies. The number of nuclei contained within each myotube was counted and the fusion index was calculated as the percentage of nuclei in myotubes over total nuclei.37 At least three random images were taken per well, and nuclei were counted using ImageJ.

The ASOs used in the studies described here were 16 nucleotides in length with a phosphorothioate backbone and three 2′‐constrained ethyl (cEt)‐modified nucleotides at both ends (3–10–3 gapmer configuration). The ASOs were either unconjugated or conjugated to a lipid (C16, palmitate) at the 5′ end via a phosphodiester linkage. For in vitro studies we had originally used conjugated ASOs, since those compounds became available first. The C16‐conjugated ASOs resulted in good activity in cells without toxicity. Unconjugated ASOs were used for in vivo studies where they successfully resulted in good activity without the need for lipid conjugation, so we decided to use the unconjugated ASOs in the in vivo setting to evaluate systemic delivery and biodistribution without unnecessary modifications. A well‐characterised control ASO which does not hybridise to any mouse mRNA sequence was included in the experiments. The oligonucleotides were synthesised and purified as previously described.62 Of the 150 ASOs targeting VCP that were initially screened for knockdown efficiency and toxicity, 10 were further evaluated in vivo for tolerability. These ASOs were administered subcutaneously to 7‐week‐old wild‐type CD‐1 male mice at a dose of 50 mg/kg weekly for 4 weeks, followed by sacrifice 72 h after the final dose (data not shown). The best performing ASOs that demonstrated the most favourable balance between efficacy and safety were chosen for further experiments. Specifically, selection was based on knockdown efficiency of VCP protein and serum biomarkers of organ toxicity.

Cell cytotoxicity upon ASO treatment was measured using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2H‐tetrazolium bromide (MTT) assay (Sigma). Briefly, 1 × 10⁴ SMPCs per well were seeded in a 96‐well plate and treated with control ASO or VCP ASO at concentrations ranging from 50 nM to 5 µM for 48 h. The treatment was conducted in serum‐reduced optiMEM medium overnight. The culture medium was then replaced with complete SMPC medium the following day. Cells were then incubated with 5 mg/mL MTT for 3 h at 37°C. A total of 100 µL of DMSO was added to each well and the culture plate placed on a shaker for 30 min to dissolve the formazan crystals, resulting in the formation of a coloured solution. The amount of formazan produced in each well was quantified by measuring the absorbance of the coloured solution at a wavelength of 570 nm. Three independent replicate measurements were collected and reported as the mean value ± standard deviation.

SMPCs were transfected with 300, 600, 900 and 1200 nM ASOs using lipofectamine 3000 (Thermofisher Scientific). Transfection was done in serum‐reduced OptiMEM medium overnight and replenished with complete SMPC medium the following day. Cells were treated with ASOs for either 3 or 7 days. For the 3‐day treatment, transfection was performed on day 1, and cells were collected on day 3. For the 7‐day treatment, transfections were conducted on days 1, 3 and 5. In parallel, cells were treated with control ASO for 7 days following the same transfection regimen. After 7 days of treatment, the cells were either fixed for immunocytochemistry or lysed for total RNA and protein extraction. To test autophagy flux, cells were incubated in serum‐free medium for 24 h to induce starvation, then treated with 10 nM BafA1 (Sigma–Aldrich; B1797) for 3 h prior to harvesting, following previously published protocol.63

Total RNA was extracted from SMPCs, ASO‐treated SMPCs and mouse tissues (quadriceps) using the Monarch Total RNA kit (New England Biolabs), and the High‐Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific) was used for cDNA synthesis. Gene expression was measured with SYBR Green PCR Master Mix, 10 ng/µL cDNA and respective primers listed in Table. Quantstudio 6 Flex Real‐Time PCR System was used to run the experiment. ΔΔCT values were calculated by comparing the treated group with its respective untreated group. S1

Preparation of protein lysates and Western blot analyses were performed as previously described.21 Briefly, cell pellets were first washed with cold phosphate buffered saline (PBS) fetal bovine serum, lysed in RIPA buffer (Sigma) with protease inhibitor cocktail for 30 min on ice and finally centrifuged ≥15 000×g for 30 min at 4°C to collect the supernatant as total soluble protein lysate. For muscle tissue (quadriceps), 30 mg of frozen tissue was first homogenised with 30–40 strokes in RIPA using a Dounce homogeniser (Wheaton Dounce Tissue Grinder, Catalog #357538), followed by shearing the tissue by passing through a 25‐gauge syringe. For further lysis, the homogenate was rotated at 4°C for ∼2 h, centrifuged at >20 000×g at 4°C for 30 min, and the supernatant was collected as soluble muscle protein lysate. The lysates were quantified using BCA assay (Thermo Fisher Scientific). A total of 20 µg of protein lysate was used for Western blot and Trans‐Blot Turbo system (Bio‐Rad Laboratories) was used to electro‐transfer proteins to 0.45‐µm polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific) and immunoblotted with primary antibodies against glyceraldehyde 3 phosphate dehydrogenase (GAPDH) (1:2500; Abcam 9485), TDP‐43 (1:2500; Abcam 190963), LC3B (1:2000; Abcam 192890), SQSTM1/p62 (1:15 000; Abcam 56416), p‐TDP‐43 (1:2500; Cosmo Bio USA: CAC‐TIP‐PTD‐M01A), ubiquitin (1:1000; Abcam 7780) and VCP (1:20 000; Abcam 11433). Secondary antibodies used were either anti‐rabbit horseradish peroxidase (HRP) (1:5000) or goat anti‐mouse HRP (1:5000).

Immunohistochemical analyses were conducted on ASO‐treated SMPCs and myotubes differentiated from SMPCs. SMPC were either cultured in matrigel coated eight‐well chamber slides (Ibidi; IbiTreat 80826) for SMPC characterisation or in four‐well chamber slides (IbiTreat 80426) for myotube differentiation study and ASO treatment on SMPCs. After the specified time, the cells were fixed in 4% paraformaldehyde. The fixed SMPCs were permeabilised with 0.2% Triton‐X, blocked with Universal Block Buffer followed by incubation at 4°C overnight with primary antibodies for MF20 (1:100; DHSB), Pax7 (1:50; DSHB), SIX1 (1:100; Novus Biologicals NPB2‐52873), TDP‐43 (1:200), LC3B (1:1000), SQSTM1/p62 (1:200), ubiquitin (1:100; Santa Cruz sc‐8017), VCP (1:500), p‐TDP‐43 (1:2000) or galectin‐3 (1:300; Santa Cruz A3A12). The cells were then washed three times with PBS and incubated with the secondary antibodies donkey anti‐rabbit Cyanine Cy™3 (Jackson ImmunoResearch; 711 165 152); or donkey anti‐mouse Alexa Fluor™ 568 (Thermo Fisher Scientific; A10042; 1:500) at room temperature for 1 h, then washed and mounted with DAPI‐containing mounting media (Vectashield; Vector Laboratories, H‐1200‐10). All images were acquired with a Zeiss LSM 900 confocal microscope (Carl Zeiss). The mean fluorescence intensity was evaluated with ImageJ. Three images were acquired for each group, and at least 30 cells were analysed.

Control non‐targeting ASO and VCP ASOs were reconstituted in PBS to a final concentration of 10 mg/mL. VCP A232E mice were injected subcutaneously with control ASO, or VCP ASO at 50 mg/kg weekly. For the safety and tolerability experiment (Figure), 10‐month‐old VCP A232E were dosed with control ASO, or VCP ASO at 50 mg/kg weekly for 3 months and compared with WT mice. Mice were regularly monitored for weight loss, alertness and activity. Weight loss of ≥15% compared with baseline was considered significant. S3

Serum was obtained from mice after 3 months of treatment with either control ASO, or VCP ASO, and used to measure ALT, AST and CK. Organ weights (liver, kidneys, spleen) were collected and analysed relative to body weight. For chronic treatment with VCP ASO to assess safety and efficacy, 6‐month‐old male VCP A232E mice were injected weekly with control ASO (n = 11) or VCP ASO (n = 11) for 3 months and assessed for motor function and compared with WT mice (n = 16). After 3 months of treatment, quadriceps muscle was collected for qPCR, Western blot and histological assessment using H&E staining. In this study, we focused on the quadriceps muscle, which exhibits prominent pathology in both VCP patients and VCP disease mouse models.49, 64, 65 As one of the most affected fast twitch predominant muscles, it provides a sensitive tissue for detecting treatment responses.

The Rotarod test was performed using a five lane Rotarod system (Med Associates Inc.) to investigate motor coordination and learning skills by measuring the ability of the mouse to stay and run on the accelerated rod. Mice are trained to walk on a rotating rod and then assessed for their ability to maintain balance as the rod accelerates from 4 to 40 RPM for a maximum time of 5 min. The test is conducted over 3 consecutive days. Each day, the mice are assessed for three trials with a recovery phase of 1–2 min between trials. As soon as a mouse falls off the rod or starts to rotate with the Rotarod without running, the timer is stopped. The latency time to fall from the rod in seconds is recorded and average from three trials and the 3‐day average is used for the analysis.

The grip strength test measures the muscle strength of the forelimbs in rodents. Grip strength is measured using a commercially available Grip Strength Meter (TSE Systems GmbH, Berlin, Germany). Each mouse is held by the base of the tail above a wire grid and gently lowered down until its front paws grasp the grid. The animal is then brought to an almost horizontal position and pulled back gently but steadily until the grip is released. Five trials testing with 1‐min intervals between trials are performed per animal. The force measurements are recorded from the five trials and averaged. The average of the 3 days of testing is used for the analysis.21

Inverted screen testing is used to assess the muscle strength of all limbs in mice. No prior training is required since the mouse has a natural motivation to hang on to the screen to avoid falling.3 Each mouse is placed on the metal wire screen for ∼1 min to allow it to adjust to the new environment. The mouse is then moved to the centre of the wire screen. The screen is flipped quickly, but gently, so the mouse is hanging upside down. The time during which the mouse is suspended upside down on the wire screen before falling is measured. The maximum time for a single trial is set at 300 s. The mouse is allowed to rest for ∼1 min before placing it on the wire again. The test is repeated for two additional trials, unless the mouse hangs on for 300 s (i.e., if the mouse hangs on for 210 s in trial 1 and 300 s in trial 2, then trial 3 was not performed). The hanging time in seconds is recorded, and the average of the three trials of testing is used for analysis.

Quadricep muscles from mice are harvested, flash frozen in 2‐methyl butane (Sigma) and embedded in optimal cutting temperature (OCT) compound. Tissue sections of 8 µm thickness are cut using a CryoStar NX50 (Epredia) microtome and subsequently stained with H&E stain following standard protocols. All images are acquired with BZ‐X800 microscope (Keyence)

Statistical analysis was performed using GraphPad Prism 9.1.2 software (GraphPad Software, Boston, MA). Data are presented as mean ± SEM. Multiple unpaired t‐tests were used for qPCR, WB densitometry quantification and immunofluorescence image analyses of VCP R155H patient in comparison with WT iPSC‐derived SMPCs. For WB analysis of WT and VCP R155H iPSC‐derived SMPCs treated with and without VCP ASO and with bafilomycin, two‐way ANOVA followed by Šídák's multiple comparisons test was used. One‐way ANOVA test followed by Dunnett's multiple comparisons test was used for qPCR and WB analyses in WT and VCP R155H iPSC‐derived SMPCs treated with different doses of VCP ASO. Two‐way repeated measures ANOVA followed by Dunnett's multiple comparisons test was used for motor testing data analysis using Rotarod, grip strength, inverted screen tests and for body weight analysis. One‐way ANOVA test followed by Dunnett's multiple comparisons test was used for qPCR and WB densitometry quantification, and organ weight analysis in treated A232E compared with WT mice and unpaired t‐test was used for central nuclei quantification. For blood toxicology and liver enzyme analyses, one‐way ANOVA followed by Fisher's least significant difference test was used. AUC analysis was performed on motor performance longitudinal data. Group‐level comparisons of AUC values were conducted using one‐way ANOVA followed by Dunnett's multiple comparisons test to evaluate treatment effects relative to control groups. The level of statistical significance was set at *p = .05, **p = .01, ***p = .001, ****p = .0001. The sample size for in vivo mouse treatment was determined based on expected changes in muscle strength over time (baseline, 3, 6 and 9 months) between treatment and control groups. Based on prior literature,21 we assumed a 15‐s change in latency to fall on the rotarod test for mutant control mice, with a standard deviation of 10 s. To detect a 10‐s difference between treatment and control groups with 80% power and a significance level of 0.0125 (adjusted for multiple timepoints), a sample size of 12 mice per group was calculated.