SUPPLEMENTARY INFORMATION

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1 SUPPLEMENTARY INFORMATION Supplementary Table 1 Supplementary Figures 1, 2, 3, 4, 5, 6, 7, 8 and legends Supplementary Video 1, 2 legends AON Sequence Position Length MW tcdna 5 -AACCTCGGCTTACCT nt OMe 5 -GGCCAAACCUCGGCUUACCU nt 6887 PMO 5 -GGCCAAACCTCGGCTTACCTGAAAT nt 8413 Supplementary Table 1 AONs targeting the mouse exon 23 donor splice site of the dystrophin gene. To ensure optimal efficiency for each chemistry, taking into account their affinity with the target and previous studies 15-16, we used 3 overlapping AONs of different lengths (M23D: tcdna (+2-13) 15-mer; 2 OMe (+2-18) 20-mer; PMO (+7-18) 25-mer). TcDNA were compared to the best published 2 OME and PMO AONs sequences published and previously used in comparison studies 17. Supplementary figures Supplementary Figure 1 - Representation of the chemical structure of tcdna as compared with DNA. 1

2 Supplementary Figure 2. Size-Exclusion Chromatography with Multi-Angle Static Light Scattering (SEC-MALS). Representative SEC-MALS analyses of tcdna, 2OMe and PMO. Analysis the different oligonucleotides at a concentration of 100 µm in PBS by size exclusion chromatography followed by in-line multi-angle light scattering. The right axis represents the molecular mass at any given point in the elution profile. The elution profiles are shown and demonstrate a significantly detectable particulate fraction only in the tcdna sample with a molecular mass of 1.58 x 10 7 Da. Fainter particulate signals can be seen for 2OMe and PMO samples; however, molecular masses cannot be determined for these signals as they are below the instrument detection limit. 2

3 Supplementary Figure 3. TcDNA AON detection over time. (a) Pharmacokinetic of tcdna in mdx mice after intravenous injection of 200 mg/kg/wk of tcdna for 4 weeks. Tissue were analysed for tcdna content by HPLC-MS/MS 2 weeks or 12 weeks after the end of the treatment. The table below displays the percentage of tcdna remaining in the tissue 12 weeks after the treatment compared to the levels detected 2 weeks after the end of the treatment. (b) The half-life of tcdna in tissue was calculated from the tissue content measured by HPLC-MS/MS 2 weeks or 12 weeks after the end of the treatment and expressed in days. 3

4 Supplementary Figure 4. Serum biomarkers levels in mdx treated mice. Serum levels of urea (a), creatinine (b), creatine kinase (CK) (c), alkaline phosphatase (ALP) (d), Aspartate aminotransferase (AST) (e) and Alanine aminotransferase (ALT) (f) were measured before and at the end of the treatment in mice treated with 200mg/kg/wk of tcdna, 2 OMe or PMO for 12 weeks. Serum CK levels are reduced in mice treated with all AONs at high dose, correlating the levels of dystrophin restoration. N=4 per group; error bars are ± SEM; *p<0.05 (t test for heteroscedastic variables). 4

5 Supplementary Figure 5. Comparison of equimolar dose of tcdna, 2 OMe and PMO on muscle function. Tibialis anterior (TA) muscles of mdx mice treated with an equimolar dose of 15 µmol of tcdna, 2 OMe or PMO were analyzed for their specific force. Specific force in tcdna treated mice is not significantly different from wild type, **p<0.01 and *p<0.05 compared to control mdx mice. The percentage of force drop reveals that tcdna treated muscles are significantly more resilient compared to 2 OMe treatment (*p<0.05) (Mann-Whitney U tests). N=4 per group; error bars ± SEM. 5

6 Supplementary Figure 6. Additional parameters of respiratory function: PIF and penh. Respiratory function was measured by whole body plethysmography in mdx mice treated with 200 mg/kg of tcdna, 2 OMe or PMO for 12 weeks (N=4 per group except for PMO N=3), compared to mdx control (N=12) and wild-type (WT) mice (N=10). Peak inspiratory flow (PIF) and enhanced pause (penh= (PEF/PIF) x Pause; Pause = (Te/RT)-1 and Te, Expiratory time; RT, time to expire 65% of the volume) are shown. Error bars are ± SEM; ***p< 0.001, **p<0.01 and #p=0.052 compared to mdx control mice (Mann-Whitney U tests). Data are shown as box plots, center lines show the medians, box limits indicate the 25th and 75th percentiles, whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles and individual data points are plotted as open circles. 6

7 Supplementary Figure 7. Stress-induced unconditioned fear. Sample individual track plots showing the distance run by mice during the 5 min testing period. Sample traces were selected to represent the average score recorded in WT and mdx control groups, and in the groups of mdx mice injected with 200 mg/kg tcdna (tcdna-200), 2 OMe (2 OMe-200) and PMO (PMO-200). The blue and red dots indicate the start and end of the tracking record, respectively. 7

8 Supplementary Figure 8. Lasting effect of tcdna treatment. The durability of the treatment was assessed by comparing treated mice 2 weeks or 12 weeks after the end of a 4-week tcdna treatment at 200 mg/kg/wk. (a-b) Detection of exon 23-skipped dystrophin mrna in mdx muscles including tibialis anterior (TA), gastrocnemius (Gas), quadriceps femoris (Quad), triceps brachialis (Tri), biceps brachialis (Bi), diaphragm (Dia) and heart 2 weeks (a) or 12 weeks (b) after the final injection. (c) Exon 23 skipping was quantified by Taqman qpcr and expressed as a percentage of total dystrophin mrna, measured by the exon 4-5 expression level, after normalization with an endogenous control. N=4 mice per group; error bars are mean ± SEM. (e-f) Western blot of total protein extracted from different muscles from treated mice 2 weeks (e) or 12 weeks (f) after the final injection, stained with the NCL-DYS μg of total protein were loaded for treated samples and 50 μg for the WT control. (g) Levels of restored dystrophin are quantified in various muscles, membranes were converted to numerical pictures by scanning, and band intensities were analyzed using the ImageJ 1.46r software. Dystrophin levels are expressed as percentage of WT levels. This suggests that tcdna are stable in cells and could be re-employed over time thus limiting the need to fill up tissues 8

9 as often as it would be required if these oligos were destroyed or tittered by their mrna target. (d-h) Another way to test durability of the treatment. Three sets of animals were treated with different tcdna dosing regimen. Set 1 for 12 weeks at 200 mg/kg/week, Set 2 for 12 weeks at 50 mg/kg/week and Set 3 for 4 weeks at 200 mg/kg/week followed by 8 weeks at 50 mg/kg/week. Muscles were collected 2 weeks after the final injection and analyzed by Taqman RT-qPCR (d) and by Western blot (h). Levels of exon skipping and dystrophin restoration obtained with the set 3 (for 4 weeks at 200mg/kg/week followed by 8 weeks at 50 mg/kg/week) are not significantly different from mice treated at 200mg/mg/kg over 12 weeks. Error bars are shown as ± SEM (N=3 per group). Supplementary Video 1- Control dko mouse. Representative decreased ambulation of a 8-week old control dko mouse. The reduced musculature considerably affects the mobility of the mice, which display a very severe dystrophic phenotype, including contracted and stiff limbs, and very pronounced kyphosis as a result of the degenerative process. Supplementary Video 2 TcDNA treated dko mouse. The dystrophic pathology of the treated mouse appears greatly improved as the animal demonstrates only a minimal kyphosis and is very mobile compared to the untreated control. 9