MEG3 , a long non-coding RNA (lncRNA), has been shown to play a critical role in regulating apoptosis. Its downregulation inhibits apoptosis in cancer cells, whereas its upregulation has been associated with cell death in both cardiovascular disease and, more recently, Alzheimer’s Disease. Here we show that MEG3 is upregulated in Myotonic Dystrophy 1 (DM1). Specifically, we show MEG3 upregulation by several-fold in DM1 human muscle cells and in two DM1 mouse models, HSA-LR and LC15. In human DM1 muscle cells we observe nuclear retention of MEG3 and an increase in its transcript diversity. Furthermore, we observe a general trend of nuclear retention in DM1 affecting lncRNAs and microRNAs (miRNAs), in contrast to mRNAs, when compared to healthy cells. This altered nuclear retention may contribute to the pathological effects of non-coding RNA dysregulation in DM1. Importantly, we demonstrate that treatment with antisense conjugates targeting the repeat expansion causing DM1, an approach currently being tested in Clinical Trials, corrects MEG3 levels in HSA-LR mice, without additional therapeutic interventions targeting MEG3 . ### Competing Interest Statement A.F.K., D.F., M.A.V. and M.J.A.W are listed as inventors on patents for the use of antisense conjugates licensed to PepGen. Medical Research Council, https://ror.org/03x94j517, MRW0147421, MRP01741X1 Muscular Dystrophy UK, 19GRO-PG36-0294 Association Francaise contre les Myopathies/AFM-Telethon, 15758

PepGen's Enhanced Delivery Oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and nuclear uptake of therapeutic oligonucleotides. PGN-EDODM1, in clinical trials for the treatment of myotonic dystrophy type 1 (DM1), is designed to bind pathogenic CUG repeat expansions in DMPK mRNA and liberate MBNL1 protein without degrading DMPK transcripts. Liberation of sequestered MBNL1 is expected to restore the normal splicing of multiple downstream transcripts; a central cause of DM1 pathology. Indeed, in vitro treatment of DM1 myotubes showed PGN-EDODM1 delivery to the nucleus, and treatment of DM1 patient myotubes with both low-CUG and high-CUG repeats demonstrated similar dose-dependent reduction of pathogenic myonuclear foci, liberation of MBNL1 from foci, and correction of mis-splicing without DMPK RNA degradation. A single intravenous (IV) dose of PGN-EDODM1 to HSALR mice resulted in dose-dependent correction of mis-splicing and improvement in myotonia. Following repeat-dosing of PGN-EDODM1 once every 4 weeks, near complete resolution of DM1 pathology including correction of mis-splicing and resolution of myotonia was observed. Toxicology studies in NHPs indicate repeat dosing with PGN-EDODM1 was generally safe and well tolerated. No persistent elevation of kidney biomarkers was observed at doses through 60 mg/kg. Additionally, there were no adverse findings in the kidney after 4 monthly doses of 60 mg/kg, nor notable hematologic or hepatic or cardiovascular effects. Currently, there are no approved therapies for DM1. Nonclinical pharmacology and safety studies with PGN-EDODM1 showed meaningful therapeutic potential on splicing correction and myotonia correction and support the ongoing Phase 1 single-ascending dose study FREEDOM-DM1 in adults with DM1.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disease that originates from an expansion of CTG microsatellites in the 30 untranslated region of the DMPK gene, thus leading to the expression of transcripts containing expanded CUG repeats (CUGexp). The pathophysiology is explained by a toxic RNA gain of function where CUGexp RNAs form nuclear aggregates that sequester and alter the function of MBNL splicing factors, triggering splicing misregulation linked to the DM1 symptoms. There is currently no cure for DM1, and most therapeutic strategies aim at eliminating CUGexp-DMPK transcripts. Here, we investigate a DMPK-promoter silencing strategy using CRISPR interference as a new alternative approach. Different sgRNAs targeting the DMPK promoter are evaluated in DM1 patient muscle cells. The most effective guides allowed us to reduce the level of DMPK transcripts and CUGexp-RNA aggregates up to 80%. The CUGexp-DMPK repression corrects the overall transcriptome, including spliceopathy, and reverses a physio-logical parameter in DM1 muscle cells. Its action is specific and restricted to the DMPK gene, as confirmed by genome-wide expression analysis. Altogether, our findings highlight DMPK-promoter silencing by CRISPRi as a promising therapeutic approach for DM1.

Objective: In vitro and in vivo evaluation of pharmacological activity via measurement of splicing changes, nuclear RNA foci, and myotonia. Background: PepGen's EDO cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides. PGN-EDODM1 is being evaluated for the treatment of DM1. PGN-EDODM1 binds to the pathogenic CUG repeat expansion in DMPK mRNA and reduces sequestration of Muscleblind (MBNL) proteins within nuclear RNA foci through a steric blocking mechanism. Release of MBNL proteins is hypothesized to correct DM1 spliceopathy; a central cause of DM1. Design/Methods: DM1 donor cells with 2,600 CTG repeats were treated with PGN-EDODM1 (1, 2, 5, 10, or 20 μM). Nuclear RNA foci profiles and splicing events were evaluated after 24 hours. PGN-EDODM1 (0, 10, 20, 30, and 50 mg/kg) was administered intravenously (IV) to wildtype (WT) or HSALR mice (DM1 murine model containing 250 CTG repeats in the HSA gene). Splicing profiles were evaluated in gastrocnemius and quadricep muscles 2-weeks post dose and myotonia was quantitatively assessed. Tissue levels of PGN-EDODM1 were analyzed. To explore the duration of pharmacological activity, PGN-EDODM1 (30 mg/kg IV) was administered to WT and HSALR mice and splicing profiles were evaluated 12 or 24 weeks post-dose. Results: The cellular model showed dose-dependent reduction in toxic RNA foci and correction of mis-splicing. In the HSALR model, a single dose resulted in high muscle concentrations of PGN-EDODM1, dose-dependent correction of mis-splicing (persisting over 24 weeks), and resolution of myotonia. PGN-EDODM1 was generally well tolerated at pharmacological doses. Conclusions: These preclinical studies demonstrate that PGN-EDODM1 reduced nuclear RNA foci and corrected mis-splicing at clinically relevant, tolerable doses with effects lasting up to 24 weeks. The HSALR mouse showed resolution of myotonia after a single PGN-EDODM1 dose. A Phase 1 clinical study in adults with DM1 will be initiated in 2023. Disclosure: Dr. Holland has received personal compensation for serving as an employee of PepGen Inc.. Dr. Holland has received intellectual property interests from a discovery or technology relating to health care. Mr. Klein has nothing to disclose. Caroline Godfrey has nothing to disclose. Dr. Svenstrup has received personal compensation for serving as an employee of PepGen, Inc. Dr. Svenstrup has stock in PepGen, Inc. Dr. Larkindale has received personal compensation for serving as an employee of PepGen. Dr. Bracegirdle has received personal compensation in the range of $100,000-$499,999 for serving as a Consultant for PepGen Inc. Dr. Furling has nothing to disclose. Dr. Goyal has received personal compensation for serving as an employee of Pepgen Inc.. Dr. Goyal has received personal compensation for serving as an employee of Wave Life Sciences. Dr. Goyal has received personal compensation in the range of $100,000-$499,999 for serving as an officer or member of the Board of Directors for Pepgen Inc.. Dr. Goyal has stock in PepGen Inc..

PepGen's enhanced delivery oligonucleotide (EDO) cell-penetrating peptide technology is engineered to optimize tissue delivery and cellular uptake of therapeutic oligonucleotides.PGN-EDODM1 is being evaluated for the treatment of myotonic dystrophy type 1 (DM1).PGN-EDODM1 binds to toxic CUG repeat expansion (CUGexp) in DMPK mRNA and acts to liberate sequestered MBNL1 protein without degrading DMPK transcript.Liberation of MBNL1 is hypothesized to restore splicing profiles of multiple downstream transcripts; a central cause of DM1 pathology.PGN-EDODM1 pharmacology was characterized using human immortalized myoblasts and the HSALR transgenic mouse model of DM1, and nonhuman primates (NHPs) were used to evaluate DMPK levels.Control and DM1 myoblasts ( ∼2600 CTGexp) were differentiated and treated with PGN EDODM1, which resulted in dose-dependent reduction in pathogenic myonuclear foci, liberation of MBNL1 from foci, and correction of mis-splicing in DM1 cells, while DMPK levels remained unchanged.Single intravenous (IV) dose of PGN-EDODM1 or control administered to HSALR mice resulted in high muscle concentrations of PGN-EDODM1, resolution of myotonia, dose-dependent correction of mis-splicing, and no significant impact on HSA expression when evaluated 2-weeks postdose.Splicing correction in HSALR mice persisted up to 24 weeks.Additional data from a repeat-dose study in HSALR mice with low PGN EDODM1 doses will be presented.Repeat IV doses of PGN-EDODM1 or control administered to NHPs every 2 weeks (10, 30, or 60mg/kg) or every 4 weeks (60mg/kg) did not result in decreases in DMPK transcript levels when evaluated 1 week after the final dose.Currently, there are no approved therapies for DM1.Nonclinical pharmacology studies with PGN-EDODM1 showed considerable therapeutic potential.Nonclinical data support the Phase 1 single-ascending dose study in adults with DM1.

Trinucleotide repeat expansions are the cause of two dozen neurodegenerative and developmental disorders. One of these, myotonic dystrophy type 1 (Steinert disease, or DM1) is due to the expansion of a CTG triplet in the 3’ UTR of the DMPK gene. We used highly specific DNA endonucleases to induce a double-strand break in the repeat tract to contract it below pathological length. Expression of a TALE Nuclease (TALEN) in human DM1 cells induced moderate CTG repeat contractions in 27% of the clones analyzed. These clones exhibited large internal deletions within the TALEN, occurring by homologous recombination between internal TALE repeats, inactivating the nuclease, and explaining its reduced efficacy. Taking advantage of the degeneracy of the genetic code, we recoded the TALEN sequence, to decrease internal redundancy and optimize codon usage. The new recoded TALEN showed increased efficacy in DM1 cells, with 68% of clones exhibiting a moderate to large contraction of the CTG repeat tract. In contrast, Staphylococcus aureus Cas9 ( Sa Cas9) was unable to contract the CTG repeat tract. In parallel, we completely sequenced to very high coverage the DM1 genome using the PacBio technology. Several clones in which the TALEN was induced were also totally sequenced. In some of them, length changes of other long CTG repeats were detected, possibly corresponding to off-target effects, all of them in introns or intergenic regions. Repeat contractions were never associated with recombination of flanking markers, suggesting that contractions most probably occur by an intra-allelic mechanism such as single-strand annealing. TALENs should now be considered as a promising gene therapy approach, not only for DM1 but also for many other microsatellite expansion disorders.### Competing Interest StatementThe authors have declared no competing interest.

Purpose Greater muscle fragility is thought to cause the exhaustion of the muscle stem cells during successive degeneration/repair cycles, leading to muscle wasting and weakness in Duchenne muscular dystrophy. Chronic voluntary exercise can partially reduce the susceptibility to contraction induced-muscle damage, i.e., muscle fragility, as shown by a reduced immediate maximal force drop following lengthening contractions, in the dystrophic mdx mice. Here, we studied the effect of Prospero-related homeobox factor 1 gene (Prox1) transfer (overexpression) using an AAV on fragility in chronically exercised mdx mice, because Prox1 promotes slower type fibres in healthy mice and slower fibres are less fragile in mdx muscle. Methods Both tibialis anterior muscles of the same mdx mouse received the transfer of Prox1 and PBS and the mice performed voluntary running into a wheel during 1 month. We also performed Prox1 transfer in sedentary mdx mice. In situ maximal force production of the muscle in response to nerve stimulation was assessed before, during and after 10 lengthening contractions. Molecular muscle parameters were also evaluated. Results Interestingly, Prox1 transfer reduced the isometric force drop following lengthening contractions in exercised mdx mice (p < 0.05 to 0.01), but not in sedentary mdx mice. It also increased the muscle expression of Myh7 (p < 0.001), MHC-2x (p < 0.01) and Trpc1 (p < 0.01), whereas it reduced that one of Myh4 (p < 0.001) and MHC-2b (p < 0.01) in exercised mdx mice. Moreover, Prox1 transfer decreased the absolute maximal isometric force (p < 0.01), but not the specific maximal isometric force, before lengthening contraction in exercised (p < 0.01) and sedentary mdx mice. Conclusion Our results indicate that Prox1 transfer increased the beneficial effect of chronic exercise on muscle fragility in mdx mice, but reduced absolute maximal force. Thus, the potential clinical benefit of the transfer of Prox1 into exercised dystrophic muscle can merit further investigation.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by Dmd mutations, resulting in the absence of dystrophin in skeletal muscle, and a greater susceptibility to damage during contraction (exercise). The current study evaluated whether voluntary exercise impacts a Dmd exon skipping and muscle physiology in a severe DMD murine model. D2-mdx mice were intramuscularly injected with an adeno-associated virus (AAV) U7 snRNA to correct Dmd reading frame, and allowed to voluntary run on a wheel for 1 month. Voluntary running did not induce muscle fiber regeneration, as indicated by the percentage of centronucleated fibers, Myh3 and Myh4 expression, and maximal force production, and thus possibly did not compromise the gene therapy approach. Voluntary running did not impact the number of viral genomes and the expression of U7 and Dmd 1 month after injection of AAV-U7 injected just before exercise initiation, but reduced the amount of dystrophin in dystrophin-expressing fibers from 80% to 65% of the muscle cross-sectional area. In conclusion, voluntary running did not induce muscle damage and had no drastic detrimental effect on the AAV gene therapy exon skipping approach in a severe murine DMD model. Moreover, these results suggest considering exercise as an additional element in the design and conception of future therapeutic approaches for DMD.

CTG repeat expansion (CTGexp) is associated with aberrant alternate splicing that contributes to cardiac dysfunction in myotonic dystrophy type 1 (DM1). Excision of this CTGexp repeat using CRISPR-Cas resulted in the disappearance of punctate ribonuclear foci in cardiomyocyte-like cells derived from DM1-induced pluripotent stem cells (iPSCs). This was associated with correction of the underlying spliceopathy as determined by RNA sequencing and alternate splicing analysis. Certain genes were of particular interest due to their role in cardiac development, maturation, and function (TPM4, CYP2J2, DMD, MBNL3, CACNA1H, ROCK2, ACTB) or their association with splicing (SMN2, GCFC2, MBNL3). Moreover, while comparing isogenic CRISPR-Cas9-corrected versus non-corrected DM1 cardiomyocytes, a prominent difference in the splicing pattern for a number of candidate genes was apparent pertaining to genes that are associated with cardiac function (TNNT, TNNT2, TTN, TPM1, SYNE1, CACNA1A, MTMR1, NEBL, TPM1), cellular signaling (NCOR2, CLIP1, LRRFIP2, CLASP1, CAMK2G), and other DM1-related genes (i.e., NUMA1, MBNL2, LDB3) in addition to the disease-causing DMPK gene itself. Subsequent validation using a selected gene subset, including MBNL1, MBNL2, INSR, ADD3, and CRTC2, further confirmed correction of the spliceopathy following CTGexp repeat excision. To our knowledge, the present study provides the first comprehensive unbiased transcriptome-wide analysis of the differential splicing landscape in DM1 patient-derived cardiac cells after excision of the CTGexp repeat using CRISPR-Cas9, showing reversal of the abnormal cardiac spliceopathy in DM1.

Background Voluntary exercise can improve skeletal muscle fragility, i.e. higher susceptibility to contraction induced-injury, as shown by a greater force drop following lengthening contractions, in the dystrophic Mdx mice as compared to healthy mice with dystrophin. This beneficial effect is related to the activation of the calcineurin activation. Unfortunately, voluntary running only partly rescued fragility, so it would be interesting to combined the effects of exercise, for example, with those of others treatments activating the calcineurin pathway and promoting slow and more oxidative fibres. This is of particular interest because slow muscle fibres are apparently less affected and genetic or pharmacological treatments promoting slow and more oxidative fibres are been shown to be beneficial in the Mdx mice. Methods Here, we tested whether voluntary exercise (1 month of running in a wheel) combined with Prospero-related homeobox factor 1 gene ( Prox1) transfer would better improve functional dystrophic features in Mdx mice as compared to the voluntary exercise single approach. Prox1 is known to promote the promotion of slow contractile gene program in healthy muscle. Results We found that Prox1 transfer promoted slower molecular and functional contractile features in both voluntary exercised and sedentary Mdx mice. However, it improved fragility only in exercised Mdx mice. Moreover, Prox1 transfer reduced absolute maximal force production by causing reduction in muscle weight in both exercised and sedentary Mdx mice. Conclusion In conclusion, our results indicate that the beneficial effects of voluntary exercise and Prox1 transfer on fragility are additive in Mdx mice.

Key points Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix. Deletion of the desmin gene in mdx mice [double knockout (DKO) mice] induces marked muscle weakness and fatigue resistance compared to mdx mice. Muscle fragility (higher susceptibility to contraction‐induced injury) was also aggravated in DKO mice compared to mdx mice. By contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy. Desmin cDNA transfer with adeno‐associated virus in newborn mdx mice reduced muscle weakness. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic muscle. AbstractDuchenne muscular dystrophy (DMD) is a severe neuromuscular disease caused by dystrophin deficiency. Desmin, similar to dystrophin, is associated with costameric structures bridging sarcomeres to the extracellular matrix that contributes to muscle function. In the present study, we attempted to provide further insight into the roles of desmin, for which the expression is increased in the muscle from the mouse mdx DMD model. We show that a deletion of the desmin gene (Des) in mdx mice [double knockout (DKO) mice, mdx:desmin–/–] induces a marked muscle weakness; namely, a reduced absolute maximal force production and increased fatigue compared to that in mdx mice. Fragility (i.e. higher susceptibility to contraction‐induced injury) was also aggravated in DKO mice compared to mdx mice, despite the promotion of supposedly less fragile muscle fibres in DKO mice, and this worsening of fragility was related to a decreased muscle excitability. Moreover, in contrast to mdx mice, the DKO mice did not undergo muscle hypertrophy, as indicated by smaller and fewer fibres, with a reduced percentage of centronucleated fibres, potentially explaining the severe muscle weakness. Notably, Desmin cDNA transfer with adeno‐associated virus in newborn mdx mice improved specific maximal force normalized to muscle weight. Overall, desmin plays important and beneficial roles in muscle wasting, performance and fragility in dystrophic mdx mice, which differ, at least in part, from those observed in healthy muscle.