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Peptide-conjugated oligonucleotides evoke long-lasting
myotonic dystrophy correction in patient-derived cells
and mice
Arnaud Klein, Miguel Varela, Ludovic Arandel, Ashling Holland, Naira
Naouar, Andrey Arzumanov, David Seoane, Lucile Revillod, Guillaume
Bassez, Arnaud Ferry, et al.
To cite this version:
Arnaud Klein, Miguel Varela, Ludovic Arandel, Ashling Holland, Naira Naouar, et al.. Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice. Journal of Clinical Investigation, American Society for Clinical Investigation, 2019, 129 (11), pp.4739-4744. �10.1172/JCI128205�. �hal-02996862�
Peptide-conjugated oligonucleotides evoke long-lasting myotonic dystrophy correction in patient-derived cells and mice.
Arnaud F. Klein1†, Miguel A. Varela2,3,4†, Ludovic Arandel1, Ashling Holland2,3,4, Naira
Naouar1, Andrey Arzumanov2,6, David Seoane2,3,4, Lucile Revillod1, Guillaume Bassez1,
Arnaud Ferry1,5, Dominic Jauvin7, Genevieve Gourdon1, Jack Puymirat7, Michael J. Gait6,
Denis Furling1#* & Matthew J. A. Wood2,3,4#*
1Sorbonne Université, Inserm, Association Institut de Myologie, Centre de Recherche en
Myologie, CRM, F-75013 Paris, France
2Department of Physiology, Anatomy and Genetics, University of Oxford, South Parks Road,
Oxford, UK
3Department of Paediatrics, John Radcliffe Hospital, University of Oxford, Oxford, UK 4MDUK Oxford Neuromuscular Centre, University of Oxford, Oxford, UK
5Sorbonne Paris Cité, Université Paris Descartes, F-75005 Paris, France
6Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue,
Cambridge, UK.
7Unit of Human Genetics, Hôpital de l'Enfant-Jésus, CHU Research Center, QC, Canada
† These authors contributed equally to the work # These authors shared co-last authorship
* To whom correspondence should be addressed
Matthew JA Wood
Department of Paediatrics
Le Gros Clark Building, South Parks Road, Oxford, OX1 3QX, United Kingdom
Tel: +44(0)1865 282840
matthew.wood@paediatrics.ox.ac.uk
Denis Furling Institute of Myology
Centre de Recherche en Myologie Sorbonne Université / Inserm /AIM G.H. Pitié-Salpétrière ; 47-83, bld de l'hôpital; 75651 Paris cedex 13, France Tel : +33 (0)1 42 16 57 07
Abstract
Antisense oligonucleotides (ASOs) targeting pathologic RNAs have shown promising therapeutic corrections for many genetic diseases including myotonic dystrophy (DM1). Thus, ASO strategies for DM1 can abolish the toxic RNA gain-of-function mechanism caused by nuclear-retained mutant transcripts containing CUG expansions (CUGexp). However, systemic use of ASOs for this muscular disease remains challenging due to poor drug distribution to skeletal muscle. To overcome this limitation, we test an arginine-rich Pip6a cell-penetrating peptide and show that Pip6a-conjugated morpholino phosphorodiamidate oligomer (PMO) dramatically enhanced ASO delivery into striated muscles of DM1 mice following systemic administration in comparison to unconjugated PMO and other ASO strategies. Thus, low dose treatment of Pip6a-PMO-CAG targeting pathologic expansions is sufficient to reverse both splicing defects and myotonia in DM1 mice and normalizes the overall disease-transcriptome. Moreover, treated DM1 patient derived muscle cells showed that Pip6a-PMO-CAG specifically targets mutant CUGexp-DMPK transcripts to abrogate the detrimental sequestration of MBNL1 splicing factor by nuclear RNA foci and consequently MBNL1 functional loss, responsible for splicing defects and muscle dysfunction. Our results demonstrate that Pip6a-PMO-CAG induces high efficacy and long-lasting correction of DM1-associated phenotypes at both molecular and functional levels, and strongly support the use of advanced peptide-conjugates for systemic corrective therapy in DM1.
Introduction
DM1, one of the most common muscular dystrophies in adults (1), is an RNA-dominant disorder caused by the expression of expanded microsatellite repeats located in the 3’ untranslated region (UTR) of the DMPK gene (2-4). Mutant transcripts containing an expanded CUG tract are retained within the nucleus as discrete foci (5, 6). Due to the high affinity for CUGexp, RNA binding factors from the MBNL family are sequestered within the CUGexp-RNA foci leading to their functional loss (7), which is a central pathophysiologic mechanism in DM1 (8). Thus, altered splicing regulatory activity of MBNL1 in striated muscles results in many alternative splicing misregulations including those in CLCN1, INSR, BIN1, DMD and
SCN5A pre-mRNAs that have been associated respectively with myotonia, insulin resistance,
muscle weakness, dystrophic process and cardiac conduction defects, all symptoms of DM1 (9-14).
Given that the toxic RNA gain-of-function mechanism in DM1 is driven by pathologic CUGexp tract within DMPK-mRNAs, therapeutic approaches using modified ASOs aimed either to degrade CUGexp-transcripts using RNase H-active ASOs (15-17) or to release sequestered MBNL1 from CUGexp-RNAs using RNase H-inactive CAGn ASOs (18-20) have demonstrated efficient reversal of DM1-associated phenotypes. However, unlike other muscle diseases like Duchenne muscular dystrophy (DMD), the integrity of the skeletal muscle fiber membrane is not compromised in DM1 resulting in poor penetration of naked PMO- or 2’-O-methyl-ASOs (21). Backbone modifications led to the design of more potent chemistries including 2’-O-methoxyethyl or 2’-4’-constrained ethyl modified residues that enhanced ASO delivery and consequently therapeutic efficacy in skeletal muscles of DM1 mice (15, 17). This approach has been tested in a human clinical trial (NCT02312011); however systemic administration of the selected naked ASO did not achieve sufficient concentration levels in skeletal muscle of patients to have marked clinical benefits. Therefore, tissue uptake remains a major challenge for ASO-therapies in DM1.
Among strategies to improve delivery of ASOs in DM1 skeletal muscles, cell-penetrating peptide conjugates offer an attractive avenue (22-24). Indeed, systemic administration of advanced Pip6a peptides conjugated with splice-switch oligonucleotides has shown promising results in DMD mice by restoring a truncated but functional dystrophin (25). Pip6a is a cell-penetrating peptide comprising a hydrophobic core region flanked on each side by arginine-rich domains containing β-alanine (B) and aminohexanoyl (X) spacers. This peptide sequence has the ability to deliver associated cargoes across the plasma and endosomal membranes and is stable to serum proteolysis (25). Here, we have investigated the potential of Pip6a conjugates to enhance RNase H-inactive CAGn-PMO delivery in skeletal muscles of DM1 mice in order to target nuclear-retained mutant CUGexp-transcripts and inhibit further toxicity associated to expanded repeats.
Results / Discussion
To test whether advanced Pip6a conjugates enhanced ASO delivery and activity in skeletal muscles of DM1 mice following systemic administration, antisense PMOs composed of a CAG7 sequence, either naked (PMO) or conjugated to Pip6a peptide (Pip6a-PMO), were injected in the tail vein of HSA-LR mice. This well-characterized DM1 mouse model expresses 250 CTG repeats in the 3’UTR of the human skeletal muscle alpha actin (HSA) gene and displays molecular as well as functional DM1-associated abnormalities (26). The skeletal muscle-specific expression of CUGexp-RNAs in HSA-LR mice leads to the formation of ribonuclear foci that sequester MBNL1 proteins resulting in splicing defects and myotonia as found in DM1 patients (11, 27).
Two weeks after a single injection of a dose of 12.5 mg/kg, mice were sacrificed and the correction of DM1-splicing defects was used as a biomarker of ASO activity. Pip6a-PMO induced a partial but significant correction of several MBNL1-dependent splicing defects (e.g.
Clcn1 exon 7a, Mbnl1 exon 5 and Atp2a1 exon 22) in HSA-LR gastrocnemius muscle whereas
no effect was observed with naked PMO at the same dose (12.5 mg/kg) or even at a much higher dose of 200 mg/kg (Figure 1A). Moreover, two and three systemic injections of Pip6a-PMO (12.5 mg/kg) led respectively to an almost complete normalization and a full correction of DM1-splicing profiles of Clcn1, Mbnl1 and Atp2a1 genes in both HSA-LR gastrocnemius and quadriceps muscles (Supplementary Figure 1 and Figure 1B). In contrast, even three injections of naked PMO at a high dose of 200 mg/kgdid not improve splicing defects. PMO concentration was also quantified in skeletal muscle of HSA-LR mice by a custom ELISA assay. Results showed PMO concentrations of >1 nM two weeks after Pip6a-PMO treatment whereas mice treated with naked PMO showed low picomolar concentrations confirming the poor uptake of naked PMO in non-renal tissue (Supplementary Figure 2). Interestingly, systemic administration of Pip6a-PMO in DM1 mice leads also to therapeutic levels of PMO-CAG in heart and diaphragm tissues that are affected in DM1 disease. Despite the restricted expression of CUGexp to the hACTA1 gene in HSA-LR mice does not allow to assess ASO therapy in cardiac tissue, our results strongly suggest that the efficient biodistribution of Pip6a conjugated PMO into striated muscles could counteracted other DM1-related symptoms including cardiac conduction defects and respiratory failure, which are the most common causes of death in DM1 patients.
To determine whether Pip6a-PMO treatment can improve muscle function, we evaluated myotonia, a DM1 hallmark characterized by delayed muscle relaxation also detectable in HSA-LR mice. As shown in Figure 1C-D, Pip6a-PMO treatment completely abolished myotonia in gastrocnemius muscles of HSA-LR mice, which is in agreement with the correction of Clcn1
exon 7a missplicing responsible for myotonia (28). In contrast, systemic injections of a control
Pip6a-PMO composed of a reverse GAC7 sequence (Pip6a-Ctrl) had no effect on either DM1-splicing defects or myotonia in HSA-LR mice (Supplementary Figure 3A-B). Altogether these results showed that a low-dose treatment of Pip6a conjugates allows efficient systemic delivery of ASO in skeletal muscle of DM1 mice. Remarkably, optimal beneficial effects at both molecular and functional levels were achieved with a cumulative dose of Pip6a-ASO that is five to ten times lower than either unconjugated or conjugated-ASOs previously described and evaluated in the same DM1 mouse model (15, 17, 22, 29).
To examine the overall effect of Pip6a-PMO on the skeletal muscle transcriptome of DM1 mice, we performed a deep paired-end RNA sequencing (RNA-seq). The principal component analysis (PCA) showed that gene expression in the gastrocnemius muscle of HSA-LR mice treated with Pip6a-PMO was shifted towards WT mice (Figure 2A). Moreover, the heatmap performed with all the transcripts having a significant change (n=3176; adj. p-value<0.1; no threshold in fold change (FC)) confirmed the global gene expression correction in treated HSA-LR mice (Figure 2B). Strikingly, the vast majority (85%) of the most significant changes observed at the gene expression level in HSA-LR mice (FC≥2 and adj. p-values<0.1; 376 transcripts) were corrected in treated mice with an average correction index of 76% (Figure 2C
and Supplementary Table 1). In addition, most of deregulated genes containing CTG repeats
≥7 in skeletal muscles were also corrected in treated HSA-LR mice (Supplementary Table 2). Transcriptomic analysis confirmed also that Pip6a-PMO treatment induced a global correction of alternative splicing misregulation measured in HSA-LR mice (adj. p-value<0.1; no threshold in fold change; Figure 2D). Thus 80% of the most deregulated splicing events in HSA-LR mice (FC≥2 and adj. p-values<0.1; 339 events) were normalized in treated mice an average correction index of 83% (Figure 2E) also leading to correction of the deregulated biological pathways (Figure 2F and Supplementary Table 3). Similar results were obtained from the quadriceps muscle of the same treated-mice (Supplementary Figure 4) indicating that the muscle transcriptome of DM1 mice could be almost normalized by Pip6a-PMO treatment targeting pathologic CUGexp. In addition, PCA and heatmap analysis (FC≥ 1.5; p-value <0.05; n=118) of the proteome assessed by label-free mass spectrometry revealed that protein expression in the quadriceps muscle of treated HSA-LR mice also tends to shift from a disease towards a wild type profile, supporting the correction of the DM1 muscle phenotype by Pip6a-PMO treatment (Supplementary Figure 5 and Supplementary Table 4).
To further determine consequences of Pip6a-PMO on CUGexp-RNA behavior, we examined nuclear RNA foci number and CUGexp-RNA levels since it has been shown previously that both features were reduced by RNase H-inactive CAGn-antisense-ASOs (18, 19). Likewise, a similar mechanism of action, which is not fully explained yet, was also observed in Pip6a-PMO treated HSA-LR mice as i) missplicing reversal due to the release of functional MBNL1 from CUGexp-RNA foci was associated with a 50% reduction in the number of myonuclei containing RNA foci in treated muscles (Figure 3A-B and Supplementary Figure 6); ii) a 60% decrease of CUGexp-RNA steady-state levels was detected following Pip6a-PMO treatment whereas control Pip6a-Ctrl treatment had no effect on CUGexp-RNA expression (Figure 3C and Supplementary Figure 3C).
Besides, the duration of Pip6a-PMO action in HSA-LR skeletal muscles was evaluated because activity and beneficial effect of nuclease-resistant ASOs could be maintained for several weeks
in vivo (30-33). Correction of splicing changes was used as a molecular biomarker and
Pip6a-PMO effect was assessed up to six months post-treatment. As observed in Figure 3D, splicing misregulation of Clcn1 exon 7a, Mbnl1 exon 5 and Atp2a1 exon 22 pre-mRNAs were completely normalized in both HSA-LR gastrocnemius and quadriceps muscles until four weeks after systemic administration. Moreover, a significant 80 to 100% of correction of these splicing defects was still measured twenty-six weeks post-treatment supporting a long-lasting activity of Pip6a-PMO, which remained nearly complete for a six-month period. Furthermore,
mild histological changes observed in liver or kidney two weeks after Pip6a-PMO injections were mostly reversed six months later (Supplementary Table 5) supporting transient and reversible side effects of Pip6a-PMO treatment.
Finally, molecular effects of Pip6a-PMO was assessed in human DM1 muscle cells carrying a large expansion (2600 repeats) and expressing within its natural context both mutant
CUGexp-DMPK and non-mutated normal-CUGexp-DMPK transcripts (34). Pip6a-PMO treatment induced the
displacement and relocation of MBNL1 from RNA foci towards the nucleoplasm as observed in WT muscle cells (Figure 4A). Moreover, MBNL1-dependent splicing defects of LDB3,
MBNL1, SOS1 and DMD pre-mRNAs that are present in DM1 differentiated muscle cells were
significantly corrected by Pip6a-PMO confirming the functional restoration of MBNL1 (Figure
4B). Similar results were obtained on another DM1 cell line carrying >1300CTG and described
previously (34) (Supplementary Figure 7). Importantly, no splicing changes were observed neither in DM1 muscle cells treated with Pip6a-Ctrl or naked PMO nor in WT muscle cells treated with Pip6a-PMO (Supplementary Figure 8). As a consequence of MBNL1 displacement from CUGexp-RNA foci, Pip6a-PMO treatment induced a 79% decrease in the number of foci per nucleus associated with an 40% increase in the number of nucleus without foci (Figure 4C-D), which was consistent with a 77% decrease of CUGexp-DMPK transcript levels in treated DM1 cells (Figure 4E-F). Remarkably, products of non-mutated DMPK alleles were not affected in DM1-treated cells supporting a specific targeting of Pip6a-PMO composed of a CAG7 sequence to mutant DMPK transcripts containing CUGexp tract.
In conclusion, our study demonstrates that Pip6a cell-penetrating-peptide improves the penetration of ASO in striated muscles of DM1 mice after systemic delivery. Thus, low dose treatment of Pip6a-conjugated PMO directed against pathogenic CUGexp repeats is sufficient to achieve an effective concentration of ASO in muscle fibers and induces an efficient and long-lasting correction of molecular and functional phenotypes in DM1 mice. Altogether these results strongly support the development of ASO-conjugates peptides for further clinical trial in DM1 as well as other microsatellite expansion disorders.
Acknowledgements
This work was supported by the Association Francaise contre les Myopathies/AFM-Telethon (grant 15758), Association Institut de Myologie, Muscular Dystrophy UK (16GRO-PG36-0091), Medical Research Council (MR/P01741X/1) and the John Fell Fund (152/058). We would like to thank Mégane Lemaitre from the Muscle Function Evaluation platform (UMS28), and C. Thornton for the MBNL1 polyclonal antibody and the HSALR mouse model.
Author contribution
Experiments were performed by A.F.K, M.A.V, L.A, A.H, D.S, A.F, L.R, and D.J. A.A produced the Pip6a-PMO. Transcriptomic analysis was performed by N.N. Proteomics data were generated and analyzed by A.H. Data were analyzed by A.F.K, M.A.V, G.B, G.G, J.P, M.J.G, M.J.W and D.F. The study was designed and written by M.J.A.W and D.F.
Competing financial interest
M.J.G. and M.J.W are co-founders of PepGen, a company committed to the enhanced peptide delivery of oligonucleotide therapeutics for treatment of neuromuscular and other diseases.
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Supplemental material
Methods
Synthesis of Peptide-PMO Conjugates. Pip6a Ac-(RXRRBRRXRYQFLIRXRBRXRB)-CO
OH was synthesized and conjugated to PMO as described previously (1). The PMO sequence targeting CUG expanded repeats (5′-CAGCAGCAGCAGCAGCAGCAG-3′) and PMO control reverse (5′-GACGACGACGACGACGACGAC-3′) were purchased from Gene Tools LLC.
Animal model and ASO injections. Experiments were carried out in the “Centre d’études
fonctionnelles” (Faculté de Médecine Sorbonne University) according to French legislation and Ethics committee approval (#1760-2015091512001083v6). HSA-LR mice are gift from Pr. Thornton. The intravenous injections were performed by single or multiple administrations via the tail vein in mice of 5 to 8 weeks of age. Doses of 12.5 mg/kg of Pip6a-PMO-CAG7 and 12.5 or 200 mg/kg of PMO were diluted in 0.9% saline and given at a volume of 5-6 µL/g of body weight. Multiple injections were done at 2 weeks apart. Myotonia was evaluated and tissues were harvested 2 weeks after the last injection. For long-term experiments, tissues were harvested 2, 4 weeks or 6 months after the last injection.
In situ myotonia / muscle relaxation measurement. The isometric contractile properties of gastrocnemius muscle were studied in situ as previously described (2). Mice were anesthetized with a solution of ketamine/xylasine(80 mg/kg and 15 mg/kg, respectively). The knee and foot were fixed with clamps and pins. The distal tendon of the gastrocnemius muscle was attached to a lever arm of a servomoteur system (305B, Dual-Mode Lever). Data were recorded and analyzed using PowerLab system (4SP, ADInstruments) and software (Chart 4, ADInstruments). The sciatic nerve (proximally crushed) was stimulated by a bipolar silver electrode using a supramaximal (10-V) square wave pulse of 0.1 ms duration. Absolute maximal isometric tetanic force (P0) was measured during isometric contractions in response to electrical stimulation (frequency of 25 to 150 Hz, train of stimulation of 500 ms). Myotonia was measured as the delay of relaxation muscle after the measure of P0.
Human cells culture and Pip6a-PMO treatment. Immortalized myoblasts from healthy
individual or DM1 patient with 2600 CTG repeats and immortalized MYOD1-inducible DM1 fibroblasts with 1300CTG repeats were previously described (3). Immortalized myoblasts were cultivated in a growth medium consisting of a mix of M199:DMEM (1:4 ratio; Life
technologies) supplemented with 20% FBS (Life technologies), 50 µg/ml gentamycin (Life technologies), 25 µg/ml fetuin, 0.5 ng/ml bFGF, 5 ng/ml EGF and 0.2 µg/ml dexamethasone (Sigma-Aldrich). Fibroblasts are cultivated in a DMEM growth medium supplemented with 15% FBS and 50 μg/mL gentamycin. Myogenic differentiation was induced by switching confluent cell cultures to DMEM medium supplemented with 5 µg/ml insulin (Sigma-Aldrich) for myoblasts and a additional 4 µg/mL of doxycycline (Sigma-Aldrich) for immortalized MYOD1-inducible fibroblasts. For treatment, WT or DM1 cells are differentiated for 4 days. Then, medium was changed with fresh differentiation medium with pip6a-PMO at a 1 µM concentration. Cells were harvested for analysis 24h after treatment.
RNA isolation, RT-PCR and qPCR analysis. For mice tissues: prior to RNA extraction,
muscles were disrupted in TriReagent (Sigma-Aldrich) using Fastprep system and Lysing Matrix D tubes (MP biomedicals). For human cells: prior to RNA extraction, cells were lysed in a proteinase K buffer (500 mM NaCl, 10 mM Tris-HCl, pH 7.2, 1.5 mM MgCl2, 10 mM
EDTA, 2% SDS and 0.5 mg/mlof proteinase K) for 45 min at 55°C. Total RNAs were isolated using TriReagent according to the manufacturer’s protocol. One microgram of RNA was reverse transcribed using M-MLV first-strand synthesis system (Life Technologies) according to the manufacturer’s instructions in a total of 20 µL. One microliter of cDNA preparation was subsequently used in a semi-quantitative PCR analysis according to standard protocol (ReddyMix, Thermo Scientific). PCR amplification was carried out for 25-35 cycles within the linear range of amplification for each gene. PCR products were resolved on 1.5-2% agarose gels, ethidium bromide-stained and quantified with ImageJ software. The ratios of exon inclusion were quantified as a percentage of inclusion relative to total intensity of isoform signals. To quantify the mRNA expression, real-time PCR was performed using a Lightcycler 480 (Roche). Reactions were performed with SYBR Green kit (Roche) and according to the manufacturer’s instructions. PCR cycles were a 15-min denaturation step followed by 50 cycles with a 94°C denaturation for 15 s, 58°C annealing for 20 s and 72°C extension for 20 s. Mouse Rrlp0 mRNA were used as standard. Data were analyzed with the Lightcycler 480 analysis software. Primers are identified in Supplementary Table 6.
Fluorescent in situ hybridization / immunofluorescence. Fluorescent in situ hybridization
(FISH) experiments were done as previously described (4) using a Cy3-labeled 2′OMe (CAG)7 probe (Eurogentec). For combined FISH-Immunofluorescence experiments, immunofluorescence staining was done after FISH last washing as described previously (5) with a rabbit polyclonal anti-MBNL1 antibody (gift from C. Thornton ; 1:2000) followed by a secondary Alexa Fluor 488-conjugated goat anti-rabbit (1:500, Life technologies) antibody. Pictures were captured using an Olympus BX60 microscope and Metamorph software (Molecular Devices). Confocal images were taken with a Nikon Ti2 microscope equipped with a motorized stage and a Yokogawa CSU-W1 spinning disk head coupled with a Prime 95 sCMOS camera (Photometrics). Images were processed with Adobe Photoshop software. Automated counting of nuclei with foci, number of foci per nuclei and/or intensity of FISH signal was done using Fiji software and custom scripts.
RNA sequencing. Total RNA from gastrocnemius and quadriceps muscles of WT, HSA-LR
and Pip6a-PMO-CAG7 treated mice were sent to the Wellcome Trust Centre for Human Genetics Sequencing Facility. After complete RNA quality control on each sample
(quantification in duplicate and RNA6000 Nano LabChip analysis on Bioanalyzer from Agilent), libraries were generated from each sample using TruSeq Stranded mRNA Library Prep Kit (Illumina), where mRNA are isolated by polyA selection, and pooled together by multiplexing them using barcoded adapters. The pooled libraries were then sequenced on Illumina's HiSeq4000 sequencing system to obtain 150bp paired end reads. Sequence alignment was provided by the Sequencing Facility where the 150bp paired end reads were trimmed and mapped on Mus musculus mm10 reference genome using HISAT software (6). Then, all bioinformatics and statistical analysis were performed using custom scripts. Differentially expressed splicing events (exon_bin) between WT, HSA-LR and Pip6a-PMO-CAG7 samples were identified using DEXSeq software(7) by (i) identifying all possible exon_bins from mm10 Gencode annotation (M12 release) and estimating exon_bin counts for each sample with the provided python scripts (ii) normalization and (iii) differential exon usage tests to quantify log2 fold changes between the different conditions and their associated p-values. Differential gene expression was computed using DESeq2 software (8) from gene counts generated by FeatureCounts tool (9). Differentially expressed exon_bins or genes were assessed when the log2 fold change was greater than 2 and the adjusted p-value for multiple comparison above 0.1.
Sample preparation for proteomic analysis. Protein was extracted from WT, HSA-LR
untreated and HSA-LR Pip6a-PMO treated (12.5 mg/kg, 3 administrations at 2 weeks intervals) from quadriceps muscle. Tissue was homogenised in lysis buffer (7 M urea, 2 M thiourea, 65 mM CHAPS, 100 mM DTT and supplemented with protease and phosphatase inhibitors) by bead extraction and incubated for 2.5 hours at 4°C. The crude extract was clarified by centrifugation at 16,000xg for 20 min. Protein samples were processed using a ReadyPrep 2-D clean up kit (BioRad) and resuspended in 6 M urea, 2 M thiourea (in 10 mM Tris-HCl, pH 8.0). Protein concentration was determined by BCA. Equalised tissue lysates containing 25 µg protein were prepared for label-free mass spectrometry analysis, as described previously (10). Protein lysates were reduced with 10 mM DTT for 30 min at 37°C and alkylated with 55 mM iodoacetamide for 30 min at room temperature in the dark. Samples were diluted with four volumes 50 mM ammonium bicarbonate and digested with 1 µg trypsin and incubated at 37°C overnight. Digestion was terminated by acidification with 2% trifluoroacetic acid (TFA) in 20% acetonitrile (3:1 (v/v) dilution). Peptides were purified using C18 spin columns (Thermo Fisher), lyophilised and stored at -20°C until mass spectrometry analysis.
Liquid chromatography-mass spectrometry analysis. Peptides were analysed by
LC-MS/MS using a Dionex Ultimate 3000 nanoUPLC and a QExactive HF mass spectrometer (Thermo Fisher). Peptides were analysed by LC-MS/MS using a Dionex Ultimate 3000 NanoUPLC system via a nano electrospray source coupled to a QExactive HF mass spectrometer (Thermo Fisher). A 500 mm x 75 µm C18 (2 µm particle size) EASY-Spray column was used to separate peptides over a 60 minute gradient of 2 – 35 % acetonitrile in 5 % DMSO, 0.1 % formic acid with a flow rate of 250 nL/min. The mass spectrometer was operated in data-dependent and positive mode. MS1 scans were acquired at a resolution of 60,000 at 200 m/z. The top 12 most abundant precursor ions with a charge of ≥ 2 were selected for HCD fragmentation.
Mass spectrometry data analysis. MS raw files were processed with MaxQuant software,
version 1.5.0.0 (11). The raw files were searched against the mouse, Mus musculus, UniProt-SwissProt FASTA database (16,997 proteins) for MS/MS based peptide identification via Andromeda. Enzyme specificity was set to trypsin, with a minimum number of seven amino acids required for peptide identification, a maximum of two missed cleavages and the FDR was set to <1% at the peptide and protein level. Default settings were used for variable and fixed modifications (variable modification; acetylation (N-terminus) and methionine oxidation; fixed modifications; carbamidomethylation). A target-decoy approach to identify peptides and proteins at an FDR <1%(12). Peptide identification was performed with the precursor mass deviation up to 4.5 ppm after time-dependent mass calibration and an allowed fragment mass deviation of 20 ppm. For label-free protein quantification, the MaxLFQ algorithm was employed for intensity determination and normalisation procedures and is fully compatible with peptide and protein separation prior to MS analysis. The minimum ratio count was set to two (13). Quantitative of high-resolution peptide profiles was based on mass-to-charge (m/z), retention time and intensity values. MaxQuant was used to calculate pairwise protein ratios by taking the median of all pairwise peptide ratios per protein. Only shared identical peptides were considered for each pairwise comparison, with a minimum number of one ratio count required for each pairwise comparison and a least-squares analysis was employed to rebuild the relative abundance profile for individual proteins. This facilitated the conservation of the total summed intensity for a protein across all the samples under analysis. ‘Match between runs’ was enabled for sample processing to maximise the number of quantification events across samples, permitting the quantification of high-resolution MS1 features that were not identified by MS2 in each single measurement (14). For matching between runs the retention time alignment window was set to 30 min and the match time window was 1 min.
Northern blot. 8-12 µg of RNA was separated on 1.2% agarose MOPS-EDTA-Sodium Acetate
(Sigma-Aldrich) gels containing 0.66 M formaldehyde (Sigma-aldrich) and transferred onto Hybond-N+ membrane (GE Healthcare) by capillary transfer with 10xSSC. Blots were hybridized with random-primed 32P-labeled (AflII-HindIII fragment of DMPK cDNA) probe in a hybridization buffer (2% SDS, 10% dextran sulfate, 1xSSPE, 100 µg/ml salmon sperm DNA, 2% Denhart's solution) at 65°C overnight. Signals were analyzed on a phosphoimager (Molecular Imager FX, Bio-Rad) and quantified using Quantity One (Bio-Rad). All values were normalized to 18S rRNA signal after hybridization with 5’-end 32P-labeled 18S rRNA-oligonucleotide probes.
ELISA based measurements of oligonucleotide concentrations in tissues.
Hybridization-Based ELISAs to determine the concentration of PMO oligonucleotides were performed as described (15) using phosphorothioate probes (Sequence (5'->3') [DIG]C*T*G*C*T*G*C*TGCTGCT*G*C*T*G*C*T*G[BIO]) double-labelled with digoxigenin and biotin. This probe was used to detect Pip6a-PMO or naked PMO concentrations in eight different tissues (brain, kidney, liver, lung, heart, diaphragm, gastrocnemius and quadriceps) from treated HSA-LR mice.
Statistical analysis. All group data are expressed as mean +/- SEM. Between groups, the
comparison was performed by Mann-Whitney test or one-way ANOVA test followed by a Newman-Keuls or Tukey post-test using Prism 6 software (GraphPad Software, Inc.). Differences between groups were considered significant when P<0.05 (*, P<0.05; **, P<0.01; ***, P<0.001, ****, P<0,0001). For proteomics data handling, normalization, statistics and annotation enrichment analysis, open-source bioinformatics platforms Perseus (version 1.5.5.3)(16) or R-studio (version 0.99.903) (R Development Core team, 2011) were employed. For pairwise comparison of proteomes a two-sides t-test statistic was employed.
Data availability: Complete raw data generated from RNA sequencing were deposited in the
Sequence Read Archive (SRA; ID PRJNA531100). Codes used for analyzing are available from the corresponding author on reasonable request.
Supplemental references
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Figure 1: Pip6a-PMO corrects molecular and functional defects in HSA-LR mice. HSA-LR mice
were injected with single or multiple doses of naked PMO-CAG7 (PMO) or Pip6a-PMO-CAG7 (Pip6a-PMO) by intravenous injection and analyzed 2 weeks after treatment. A) Quantification of splicing correction induced by single 12.5 mg/kg dose of Pip6a-PMO-CAG7 treatment, 12.5 mg/kg or 200 mg/kg dose of PMO-CAG7 treatment in gastrocnemius of treated mice (n=4 for WT mice, n=10 for HSA-LR, n=4 for PMO mice and n=6 for Pip6a-PMO-CAG7). B) Representative RT-PCR results and quantification of alternative splicing profiles in gastrocnemius (gast.) and quadriceps (quad.) of HSA-LR mice treated with three injections of PMO-CAG7 at 200 mg/kg or Pip6a-PMO-CAG7 at 12.5 mg/kg (n=7 for WT mice, n=16 for HSA-LR, n=4 for PMO mice and n=8 for Pip6a-PMO-CAG7). C) Representative maximal force/time curves obtained by in situ force measurements of Pip6a-PMO-CAG7 treated mice (3 x 12.5 mg/kg) compared to WT and untreated HSA-LR mice. D) Correction of myotonia (measured as the area under the force/time curve during relaxation after maximal muscle contraction) in Pip6a-PMO injected HSA-LR mice (n=6 for WT mice, n=7 for HSA-LR and n=8 for Pip6a-PMO-CAG7). Data are expressed as mean
+/- SEM, except for D: box 25-75 percentile; whiskers min to max; central black line indicates the mean. Statistics: One-way ANOVA with Newman-Keuls post-test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n.s., non-significant.
Figure 2: Treatment with Pip6a-PMO normalizes global transcriptome at both expression and splicing levels. Transcriptomic analysis by RNA sequencing was performed on total RNA
of the gastrocnemius muscle of treated mice compared to HSA-LR and wild-type mice (n=3).
A-B) Principal component analysis (A) and heatmap graphic (B) of all the significantly
expressed transcripts (adj. p-value>0.1) of gastrocnemius muscle from either WT, HSA-LR or Pip6a-PMO-CAG7 (Pip6a-PMO) treated mice reveal a global correction of genes expression profile with Pip6a-PMO treatment. C) Treatment with Pip6a-PMO induces the correction of the expression profile in gastrocnemius muscle of treated mice (n=376; FC≥2, adj. p-value<0.1): 85.5% of transcripts return to FC<2 with an average correction index of 76%; 8% of transcripts remains at FC>2 but with correction index >20%; 6.5% of transcripts are not corrected. D) Heatmap graphic of all significant deregulated exon_bin (normalized counts) reveals a global correction of missplicing events with Pip6a-PMO treatment. E) Treatment with Pip6a-PMO induces a correction of alternative splicing profiles in gastrocnemius muscle of treated mice (n=339 splicing events; FC≥2, adj. p-value<0.1): 80% of events return to FC<2 with an average correction index of 83%; 11% remains at FC>2 but with correction index>20%; 8% are not corrected. F) Number of genes and p-values for significantly enriched Biological Process_GO_terms and Pathways in the set of HSA-LR/WT missplicing events using DAVID and Toppgene tools.
Figure 3: Treatment with Pip6a-PMO normalizes DM1-specific phenotype. HSA-LR mice
were injected in tail vein with three 12.5 mg/kg doses of Pip6a-PMO-CAG7 (Pip6a-PMO), and analyzed 2 weeks after treatment or at the indicated time. A) Representative pictures of gastrocnemius muscle section stained for CUGexp foci (FISH, red), MBNL1 (green), fibers membrane (WGA, white) and nucleus (Hoechst, blue) (scale bar : 50µm). B) Quantification of the number of nuclei with foci in gastrocnemius muscle sections of HSA-LR Pip6a-PMO treated mice (n=7 for HSA-LR and n=8 for Pip6a-PMO-CAG7). C) Quantification of HSA transcripts levels by qRT-PCR in HSA-LR treated mice (n=12 for HSA-LR and n=8 for Pip6a-PMO-CAG7). D) Quantification of splicing correction induced by Pip6a-PMO treatment in gastrocnemius (gast.) and quadriceps (quad.) at 2 weeks, 4 weeks and 6 months after injection (n=16 for HSA-LR and n=4 for each time point of Pip6a-PMO-CAG7). Data are
expressed as mean +/- SEM. Statistics B: Mann-Whitney test; C and D: One-way ANOVA with Newman-Keuls post-test; *, P<0.05; **, P<0.01; ***, P<0.001; n.s., non-significant.
Figure 4: Pip6a-PMO corrects DM1-specific molecular symptoms in DM1 muscle cells.
Four days differentiated immortalized DM1 myoblasts (2600 CTG repeats) are treated with Pip6a-PMO-CAG7 (Pip6a-PMO) at 1 µM and analyzed after 24h. A) FISH (CAG probe, red) /immuno-fluorescence (MBNL1, green) on DM1 or WT differentiated cells treated with Pip6a-PMO (scale bar : 10 µm). B) Quantification of splicing corrections, using RT-PCR induced by Pip6a-PMO treatment in immortalized DM1 differentiated muscle cells (WT n=4; DM1 and Pip6a-PMO n=7). C) Quantification of mean number of foci per nucleus in treated DM1 differentiated cells (n=4; >500 nucleus per n). D) Quantification of mean number of nucleus with foci in treated DM1 differentiated cells (n=4; >500 nucleus per n).
E) Visualization of both norm and CUGexp DMPK transcripts by Northern blot using a DMPK
probe. F) Quantification of the levels of mutDMPK / normDMPK analyzed by Northern blot (n=4). Data are expressed as mean +/- SEM. Statistics: B and F: One-way ANOVA with
Newman-Keuls post-test; C and D: Mann-Whitney test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001; n.s., non-significant.
Supplementary figure 1: Dose-dependent effect of Pip6a-PMO on alternative splicing correction in HSA-LR mice. Quantification of the alternative splicing correction in HSA-LR
mice treated with multiple intravenous systemic injections of Pip6a-PMO-CAG7 (pip6a-PMO) at the dose of 12.5 mg/kg and sacrificed after 2 weeks (n=7 fot WT, n=10 for HSA-LR, n=6 for 1 inj., n=4 for 2 inj. and 3 inj.). Data are expressed as mean +/- SEM. Statistics:
One-way ANOVA with Newman-Keuls post-test; *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.
Supplementary figure 2: Evaluation of Pip6a-PMO biodistribution reveals optimal delivery to critically affected tissues in DM1. PMOs were detected by a custom ELISA assay using
probes labelled with digoxigenin and biotin after 3 intravenous systemic injections of Pip6a-PMO-CAG7 (Pip6a-PMO) at 12.5 mg/kg or 3x200 mg/kg of naked Pip6a-PMO-CAG7 (Naked PMO) in HSA-LR mice. Two weeks after the Pip6a-PMO injections the concentration of PMO in muscle tissues was still >1nM vs the low pM detected after naked PMO injections (despite of the >20-fold difference in molarity of naked PMO vs Pip6a-PMO treatments) (n=4). Data are
Supplementary figure 3: Treatment with Pip6a-Ctrl (GAC7) has no effect in HSA-LR mice.
HSA-LR mice are treated with 3 systemic IV injections of Pip6a-PMO-GAC7 (Pip6a-ctrl) at 12.5 mg/kg and sacrificed 2 weeks after. Treatment with Pip6a-ctrl has no effect on A) the alternative splicing profiles of Clcn1, Mbnl1 and Serca1 as analyzed by RT-PCR (n=7 for HSA-LR and n=4 for Pip6a-PMO-ctrl), B) in the levels of myotonia of the gastrocnemius as measured as the area under the force/time curve during relaxation after maximal muscle contraction (n=6 for HSA-LR and n=8 for Pip6a-PMO-ctrl) and C) in levels of CUGexp HSA transcripts as determined by real-time PCR (n=12 for HSA-LR and n=4 for Pip6a-PMO-ctrl).
Data are expressed as mean +/- SEM. Statistics: A, C: One-way ANOVA with Newman-Keuls post-test; B: Mann-Whitney test; *, P<0.05; **, P<0.01; ***, P<0.001.
Supplementary figure 4: Treatment with Pip6a-PMO normalizes global transcriptome at both expression and splicing levels. Transcriptomic analysis by RNA sequencing was
performed on total RNA of the quadriceps muscle of treated mice compared to HSA-LR and wild-type mice (n=3). A-B) Principal component analysis (A) and heatmap graphic (bB) of all the significantly expressed transcripts (adj. p-value<0.1) of quadriceps muscle from either WT, HSA-LR or Pip6a-PMO-CAG7 (Pip6a-PMO) treated mice reveal a global correction of genes expression profile with Pip6a-PMO treatment. C) Treatment with Pip6a-PMO induces the correction of the expression profile in quadriceps muscle of treated mice (n=588; FC≥2, adj. p-value<0.1): 84% of transcripts return to FC<2 with an average correction index of 76% ; 10% of transcripts remains at FC>2 but with correction index >20%; 6% of transcripts are not corrected. D) Heatmap graphic of all significant deregulated exon_bin (normalized counts) reveals a global correction of missplicing events with Pip6a-PMO treatment. E) Treatment with Pip6a-PMO induces a correction of alternative splicing profiles in quadriceps muscle of treated mice (n=242 splicing events; FC≥2, adj. p-value<0.1): 82% of events return to FC<2 with an average correction index of 83%; 4% remains at FC>2 but with correction index >20%; 14% are not corrected. F) Number of genes and p-values for significantly enriched Biological Process_GO_terms and Pathways in the set of HSA-LR/WT missplicing events using DAVID and Toppgene tools.
Supplementary figure 5: Treatment with Pip6a-PMO normalizes the DM1 proteomic profile of HSA-LR mice.
Supplementary figure 5: Treatment with Pip6a-PMO normalizes the DM1 proteomic profile of HSA-LR mice. Quadriceps muscle samples from WT, HSA-LR and Pip6a-PMO-CAG7 (Pip6a-PMO)
treated HSA-LR mice were analyzed by label-free mass spectrometry. A) Correlation plots for quadriceps muscle from WT, HSA-LR and Pip6a-PMO treated HSA-LR mice (n=3 biological replicates per group). Each plot compares the quantitative intensity measurement of LC-MS features between two replicates. The Pearson correlation r-values demonstrate high reproducibility between and across all data sets, with a minimum r-value of 0.905 and a maximum of 0.963. B) Principal component analysis (PCA) illustrates a clear separation of the WT (blue), HSA-LR (red) and Pip6a-PMO treated HSA-LR (green) proteome, based on component 1 and component 2, which account for 31.1% and 16.2% of variability, respectively. PCA analysis illustrates Pip6a-PMO treated HSA-LR mice have a protein profile shift from a HSA-LR profile towards a WT profile, indicating Pip6a-PMO treatment can induce protein normalisation. C) Heatmap of 118 significant differentially expressed proteins from WT, HSA-LR and Pip6a-PMO treated HSA-LR mice. Differentially expressed proteins were analysed by hierarchical clustering of the z-scored normalised LFQ intensities (log2) across all experimental groups. Red indicates increased protein abundance and blue indicates decreased protein abundance. D) Table with the top 6 increased and decreased proteins from quadriceps muscle from HSA-LR versus WT mice as revealed by label-free mass spectrometry analysis. E) Dot plots of specific proteins of interest identified from outlining the dynamic change of normalised LFQ intensities (log2) from quadriceps muscle samples from WT, HSA-LR and Pip6a-PMO treated HSA-LR mice. Data are
expressed as mean +/- SEM. Statistics: One-way ANOVA and Bonferroni post hoc test; n.s., not significant; *, P≤0.05; **, P≤0.01; ***, P≤0.001.
Supplementary figure 6: Treatment with Pip6a-PMO reduces the foci level in the muscles of HSA-LR mice. HSA-LR mice were treated with 3 systemic injections of Pip6a-PMO-CAG7
(Pip6a-PMO) at a 12.5 mg/kg dose and sacrificed 2 weeks after treatment. A and C) Representative pictures of non-treated HSA-LR gastrocnemius sections stained for CUGexp-RNA foci (CAG probe, red), fibers membrane (WGA, green) and nucleus (Hoechst, blue). B and D) Same fields as in A and C where only the FISH signal is shown. E and G) Representative pictures of Pip6a-PMO treated HSA-LR gastrocnemius stained for CUGexp-RNA foci (CAG probe, red), fibers membrane (WGA, green) and Hoechst (blue). F and H) Same fields as in E and G where only the FISH signal is shown. Scale bar: 50 µm.
Supplementary Figure 7: Pip6a-PMO corrects DM1-specific molecular symptoms in trans-differentiated muscle cells. Four days trans-trans-differentiated immortalized DM1 fibroblasts
(1300 CTG repeats) are treated with Pip6a-PMO-CAG7 (Pip6a-PMO) at 1 µM and analyzed after 24h. A) FISH (CAG probe, red) /immuno-fluorescence (MBNL1, green) on DM1 or WT differentiated cells treated with Pip6a-PMO (Scale bar: 10 µm). B) Quantification of mean number of foci per nucleus in treated DM1 differentiated cells (n=3; >300 nucleus per n). C) Quantification of splicing corrections, using RT-PCR, induced by Pip6a-PMO treatment in immortalized DM1 trans-differentiated muscle cells (n=5). Data are expressed as mean
+/-SEM. Statistics: B, t-test ; C, One-way ANOVA with Newman-Keuls post-test; *, P<0.05; **, P<0.01; ***, P<0.001 ; ns: non-significant.
Supplementary figure 8: Effect of Pip6a-PMO on alternative splicing profiles is specific to DM1 cells and CAG antisense sequence. A) Quantification of splicing changes of LDB3, MBNL1, SOS1, DMD transcripts in 4 days differentiated immortalized DM1 (2600 CTG repeats)
myoblasts treated with Pip6a-PMO-Control (Pip6a-ctrl) at 1 µM and analyzed after 24 h (n=4).
B) Quantification of splicing changes of LDB3, MBNL1, SOS1, DMD transcripts in 4 days
differentiated immortalized WT myoblasts treated with Pip6a-PMO-CAG7 (Pip6a-PMO) at 1 µM and analyzed after 24 h (n=4). C) Quantification of splicing changes of LDB3, MBNL1, SOS1,
DMD transcripts in 4 days differentiated immortalized DM1 (2600 CTG repeats) myoblasts
treated with unconjugated PMO-CAG (PMO) at 1 µM and analyzed after 24 h (n=3). Data are
expressed as mean +/- SEM. Statistics: One-way ANOVA with Newman-Keuls post-test; ns: non-significant.
