Supplementary Material Peptide-Conjugated Oligonucleotides Evoke Long-Lasting Myotonic Dystrophy Correction in Patient-Derived C
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Supplementary material 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 Arzumanov2,5, David Seoane2,3,4, Lucile Revillod1, Guillaume Bassez1, Arnaud Ferry1,6, Dominic Jauvin7, Genevieve Gourdon1, Jack Puymirat7, Michael J. Gait5, 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 5Medical Research Council, Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK 6Sorbonne Paris Cité, Université Paris Descartes, F-75005 Paris, France 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 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 servomotor 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. Cell 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 murine DMSXL MYOD1- inducible fibroblasts with 1500 CTG repeats in the 3’UTR of the human DMPK gene are derived from DMSXL mice (4) and were modified as previously described (5). 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 the trans-differentiation of 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/ml of 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 (6) 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 (7) 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. Single molecule FISH (smFISH) was performed as previously described (8). Briefly cells are incubated with a mix a 24 human DMPK-specific DMA probes which are hybridized to a Cy3- labelled universal FLAP oligonucleotide (Supplementary table 6). 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 (9). 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(10) 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 (11) from gene counts generated by FeatureCounts tool (12). 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 (13). 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.