Targeted Antisense Oligonucleotide-Mediated Skipping of Murine Postn Exon 17 Partially Addresses Fibrosis in D2.mdx Mice.

Periostin, a multifunctional 90 kDa protein, plays a pivotal role in the pathogenesis of fibrosis across various tissues, including skeletal muscle. It operates within the transforming growth factor beta 1 (Tgf-ß1) signalling pathway and is upregulated in fibrotic tissue. Alternative splicing of Periostin's C-terminal region leads to six protein-coding isoforms. This study aimed to elucidate the contribution of the isoforms containing the amino acids encoded by exon 17 (e17+ Periostin) to skeletal muscle fibrosis and investigate the therapeutic potential of manipulating exon 17 splicing. We identified distinct structural differences between e17+ Periostin isoforms, affecting their interaction with key fibrotic proteins, including Tgf-ß1 and integrin alpha V. In vitro mouse fibroblast experimentation confirmed the TGF-ß1-induced upregulation of e17+ Periostin mRNA, mitigated by an antisense approach that induces the skipping of exon 17 of the Postn gene. Subsequent in vivo studies in the D2.mdx mouse model of Duchenne muscular dystrophy (DMD) demonstrated that our antisense treatment effectively reduced e17+ Periostin mRNA expression, which coincided with reduced full-length Periostin protein expression and collagen accumulation. The grip strength of the treated mice was rescued to the wild-type level. These results suggest a pivotal role of e17+ Periostin isoforms in the fibrotic pathology of skeletal muscle and highlight the potential of targeted exon skipping strategies as a promising therapeutic approach for mitigating fibrosis-associated complications.

Systemic Pharmacotherapeutic Treatment of the ACTA1-MCM/FLExDUX4 Preclinical Mouse Model of FSHD.

Aberrant expression of the double homeobox 4 (DUX4) gene in skeletal muscle predominantly drives the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD). We recently demonstrated that berberine, an herbal extract known for its ability to stabilize guanine-quadruplex structures, effectively downregulates DUX4 expression in FSHD patient-derived myoblasts and in mice overexpressing exogenous DUX4 after viral vector-based treatment. Here, we sought to confirm berberine's inhibitory efficacy on DUX4 in the widely used FSHD-like transgenic mouse model, ACTA1-MCM/FLExDUX4, where DUX4 is induced at pathogenic levels using tamoxifen. Animals repeatedly treated with berberine via intraperitoneal injections for 4 weeks exhibited significant reductions in both mRNA and protein levels of DUX4, and in mRNA expression of murine DUX4-related genes. This inhibition translated into improved forelimb muscle strength and positive alterations in important FSHD-relevant cellular pathways, although its impact on muscle mass and histopathology was less pronounced. Collectively, our data confirm the efficacy of berberine in downregulating DUX4 expression in the most relevant FSHD mouse model. However, further optimization of dosing regimens and new studies to enhance the bioavailability of berberine in skeletal muscle are warranted to fully leverage its therapeutic potential for FSHD treatment.

Systemic antisense therapeutics inhibiting DUX4 expression ameliorates FSHD-like pathology in an FSHD mouse model.

Aberrant expression of the double homeobox 4 (DUX4) gene in skeletal muscle causes muscle deterioration and weakness in Facioscapulohumeral muscular dystrophy (FSHD). Since the presence of a permissive pLAM1 polyadenylation signal is essential for stabilization of DUX4 mRNA and translation of DUX4 protein, disrupting the function of this structure can prevent expression of DUX4. We and others have shown promising results using antisense approaches to reduce DUX4 expression in vitro and in vivo following local intramuscular administration. Here we demonstrate that further development of the antisense chemistries enhances in vitro antisense efficacy. The optimal chemistry was conjugated to a cell-penetrating moiety and was systemically administered into the tamoxifen-inducible Cre-driver FLExDUX4 double-transgenic mouse model of FSHD. After four weekly treatments, mRNA quantities of DUX4 and target genes were reduced by 50% that led to 12% amelioration in muscle atrophy, 52% improvement in in situ muscle strength, 17% reduction in muscle fibrosis and prevention of shift in the myofiber type profile. Systemic DUX4 inhibition also significantly improved the locomotor activity and reduced the fatigue level by 22%. Our data demonstrate that the optimized antisense approach has potential of being further developed as a therapeutic strategy for FSHD.

Microdystrophin Gene Addition Significantly Improves Muscle Functionality and Diaphragm Muscle Histopathology in a Fibrotic Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease affecting 1:5000 newborn males. No cure is currently available, but gene addition therapy, based on the adeno-associated viral (AAV) vector-mediated delivery of microdystrophin transgenes, is currently being tested in clinical trials. The muscles of DMD boys present significant fibrotic and adipogenic tissue deposition at the time the treatment starts. The presence of fibrosis not only worsens the disease pathology, but also diminishes the efficacy of gene therapy treatments. To gain an understanding of the efficacy of AAV-based microdystrophin gene addition in a relevant, fibrotic animal model of DMD, we conducted a systemic study in juvenile D2.mdx mice using the single intravenous administration of an AAV8 system expressing a sequence-optimized murine microdystrophin, named MD1 (AAV8-MD1). We mainly focused our study on the diaphragm, a respiratory muscle that is crucial for DMD pathology and that has never been analyzed after treatment with AAV-microdystrophin in this mouse model. We provide strong evidence here that the delivery of AAV8-MD1 provides significant improvement in body-wide muscle function. This is associated with the protection of the hindlimb muscle from contraction-induced damage and the prevention of fibrosis deposition in the diaphragm muscle. Our work corroborates the observation that the administration of gene therapy in DMD is beneficial in preventing muscle fibrosis.

Evaluation of the dystrophin carboxy-terminal domain for micro-dystrophin gene therapy in cardiac and skeletal muscles in the DMDmdx rat model.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the gene encoding dystrophin. Gene therapy using micro-dystrophin (MD) transgenes and recombinant adeno-associated virus (rAAV) vectors hold great promise. To overcome the limited packaging capacity of rAAV vectors, most MD do not include dystrophin carboxy-terminal (CT) domain. Yet, the CT domain is known to recruit α1- and ß1-syntrophins and α-dystrobrevin, a part of the dystrophin-associated protein complex (DAPC), which is a signaling and structural mediator of muscle cells. In this study, we explored the impact of inclusion of the dystrophin CT domain on ΔR4-23/ΔCT MD (MD1), in DMDmdx rats, which allows for relevant evaluations at muscular and cardiac levels. We showed by LC-MS/MS that MD1 expression is sufficient to restore the interactions at a physiological level of most DAPC partners in skeletal and cardiac muscles, and that inclusion of the CT domain increases the recruitment of some DAPC partners at supra-physiological levels. In parallel, we demonstrated that inclusion of the CT domain does not improve MD1 therapeutic efficacy on DMD muscle and cardiac pathologies. Our work highlights new evidences of the therapeutic potential of MD1 and strengthens the relevance of this candidate for gene therapy of DMD.

G-quadruplex ligands mediate downregulation of DUX4 expression.

Abnormal DUX4 expression in skeletal muscles plays a key role in facioscapulohumeral muscular dystrophy (FSHD) pathogenesis, although the molecular mechanisms regulating DUX4 expression are not fully defined. Using bioinformatic analysis of the genomic DUX4 locus, we have identified a number of putative G-quadruplexes (GQs) forming sequences. Their presence was confirmed in synthetic oligonucleotiode sequences derived from the enhancer, promoter and transcript of DUX4 through circular dichroism and nuclear magnetic resonance analysis. We further examined the binding affinity of a naturally occurring GQ stabilizing compound, berberine, to these non-canonical genetic structures using UV-Vis and fluorescence spectroscopy. Subsequent in vitro study in FSHD patient myoblasts indicated that berberine treatment reduced DUX4 expression and also expression of genes normally switched on by DUX4. Further investigation in a mouse model overexpressing exogenous DUX4 confirmed the therapeutic effects of berberine in downregulating DUX4 protein expression, inhibiting muscle fibrosis, and consequently rescuing muscle function. Our data demonstrate for the first time that GQs are present in the DUX4 locus and that the GQ interactive ligand reduces DUX4 expression suggesting potential role of GQs in FSHD pathogenesis. Our work provides the basis of a novel therapeutic strategy for the treatment of FSHD.

Functional muscle recovery following dystrophin and myostatin exon splice modulation in aged mdx mice.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 3500-5000 newborn boys. It is due to mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibres and their replacement with fibro-adipogenic tissue. Out-of-frame mutations in the DMD gene can be modified by using antisense oligonucleotides (AONs) to promote skipping of specific exons such that the reading frame is restored and the resulting protein produced, though truncated, is functional. We have shown that AONs can also be used to knock down myostatin, a negative regulator of muscle growth and differentiation, through disruption of the transcript reading frame, and thereby enhance muscle strength. In young mdx mice, combined dystrophin and myostatin exon skipping therapy greatly improved DMD pathology, compared to the single dystrophin skipping approach. Here we show that in aged (>15-month-old) mdx mice, when the pathology is significantly more severe and more similar to the one observed in DMD patients, the effect of the combined therapy is slightly attenuated but still beneficial in improving the disease phenotype. These results confirm the beneficial outcome of the combination approach and support its translation into DMD clinical trials.

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.

Antisense targeting of 3' end elements involved in DUX4 mRNA processing is an efficient therapeutic strategy for facioscapulohumeral dystrophy: a new gene-silencing approach.

Defects in mRNA 3'end formation have been described to alter transcription termination, transport of the mRNA from the nucleus to the cytoplasm, stability of the mRNA and translation efficiency. Therefore, inhibition of polyadenylation may lead to gene silencing. Here, we choose facioscapulohumeral dystrophy (FSHD) as a model to determine whether or not targeting key 3' end elements involved in mRNA processing using antisense oligonucleotide drugs can be used as a strategy for gene silencing within a potentially therapeutic context. FSHD is a gain-of-function disease characterized by the aberrant expression of the Double homeobox 4 (DUX4) transcription factor leading to altered pathogenic deregulation of multiple genes in muscles. Here, we demonstrate that targeting either the mRNA polyadenylation signal and/or cleavage site is an efficient strategy to down-regulate DUX4 expression and to decrease the abnormally high-pathological expression of genes downstream of DUX4. We conclude that targeting key functional 3' end elements involved in pre-mRNA to mRNA maturation with antisense drugs can lead to efficient gene silencing and is thus a potentially effective therapeutic strategy for at least FSHD. Moreover, polyadenylation is a crucial step in the maturation of almost all eukaryotic mRNAs, and thus all mRNAs are virtually eligible for this antisense-mediated knockdown strategy.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing.

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein.

Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases.

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤ 0.1% to ≈ 6%) with that of homologous gene targeting (≤ 0.1% to ≈ 15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Expression of the Pro-Fibrotic Marker Periostin in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is characterised by fibrotic tissue deposition in skeletal muscle. We assessed the role of periostin in fibrosis using mdx mice, an established DMD murine model, for which we conducted a thorough examination of periostin expression over a year. RNA and protein levels in diaphragm (DIA) muscles were assessed and complemented by a detailed histological analysis at 5 months of age. In dystrophic DIAs, periostin (Postn) mRNA expression significantly exceeded that seen in wildtype controls at all timepoints analysed, with the highest expression at 5 months of age (p < 0.05). We found Postn to be more consistently highly expressed at the earlier timepoints compared to established markers of fibrosis like transforming growth factor-beta 1 (Tgf-ß1) and connective tissue growth factor (Ctgf). Immunohistochemistry confirmed a significantly higher periostin protein expression in 5-month-old mdx mice compared to age-matched healthy controls (p < 0.01), coinciding with a significant fibrotic area percentage (p < 0.0001). RT-qPCR also indicated an elevated expression of Tgf-ß1, Col1α1 (collagen type 1 alpha 1) and Ctgf in mdx DIAs compared to wild type controls (p < 0.05) at 8- and 12-month timepoints. Accordingly, immunoblot quantification demonstrated elevated periostin (3, 5 and 8 months, p < 0.01) and Tgf-ß1 (8 and 12 months, p < 0.001) proteins in the mdx muscle. These findings collectively suggest that periostin expression is a valuable marker of fibrosis in this relevant model of DMD. They also suggest periostin as a potential contributor to fibrosis development, with an early onset of expression, thereby offering the potential for timely therapeutic intervention and its use as a biomarker in muscular dystrophies.

Systemic Delivery of a Monoclonal Antibody to Immunologically Block Myostatin in the A17 Mouse Model of OPMD.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset rare muscle disease affecting approximately 1 in 80,000 individuals worldwide. However, it can affect as much as 1:600 individuals in some populations due to a strong founder effect. The muscle pathology is characterized by progressive eyelid drooping (ptosis), swallowing difficulties (dysphagia), and limb weakness at later stages of disease progression. The genetic defect is associated with significant fibrotic deposition and atrophy in affected muscles. No treatments are available to cure the disease. Only surgical techniques to correct ptosis and swallowing are currently possible, though they carry a risk of recurrence. Myostatin is a negative regulator of muscle growth, and several strategies to downregulate its expression have been developed with the aim of improving muscle mass and strength in muscular pathologies. We recently showed that weekly systemic treatment of the A17 murine model of OPMD with a monoclonal antibody for myostatin improves body and muscle mass, increases muscle strength, and reduces muscle fibrosis. Here, we describe the methodology for repeated intraperitoneal delivery of myostatin antibody in the murine model. Furthermore, we detail the most relevant analyses to assess histopathological and functional improvements of this treatment in this mouse model.

Novel Metabolomic Approach for Identifying Pathology-Specific Biomarkers in Rare Diseases: A Case Study in Oculopharyngeal Muscular Dystrophy (OPMD).

The identification of metabolomic biomarkers relies on the analysis of large cohorts of patients compared to healthy controls followed by the validation of markers in an independent sample set. Indeed, circulating biomarkers should be causally linked to pathology to ensure that changes in the marker precede changes in the disease. However, this approach becomes unfeasible in rare diseases due to the paucity of samples, necessitating the development of new methods for biomarker identification. The present study describes a novel approach that combines samples from both mouse models and human patients to identify biomarkers of OPMD. We initially identified a pathology-specific metabolic fingerprint in murine dystrophic muscle. This metabolic fingerprint was then translated into (paired) murine serum samples and then to human plasma samples. This study identified a panel of nine candidate biomarkers that could predict muscle pathology with a sensitivity of 74.3% and specificity of 100% in a random forest model. These findings demonstrate that the proposed approach can identify biomarkers with good predictive performance and a higher degree of confidence in their relevance to pathology than markers identified in a small cohort of human samples alone. Therefore, this approach has a high potential utility for identifying circulating biomarkers in rare diseases.

Antisense-induced myostatin exon skipping leads to muscle hypertrophy in mice following octa-guanidine morpholino oligomer treatment.

Myostatin is a negative regulator of muscle mass, and several strategies are being developed to knockdown its expression to improve muscle-wasting conditions. Strategies using antimyostatin-blocking antibodies, inhibitory-binding partners, signal transduction blockers, and RNA interference system (RNAi)-based knockdown have yielded promising results and increased muscle mass in experimental animals. These approaches have, however, a number of disadvantages such as transient effects or adverse immune complications. We report here the use of antisense oligonucleotides (AOs) to manipulate myostatin pre-mRNA splicing and knockdown myostatin expression. Both 2'O-methyl phosphorothioate RNA (2'OMePS) and phosphorodiamidate morpholino oligomers (PMO) led to efficient exon skipping in vitro and in vivo and knockdown of myostatin at the transcript level. The substantial myostatin exon skipping observed after systemic injection of Vivo-PMO into normal mice led to a significant increase in soleus muscle mass as compared to the controls injected with normal saline suggesting that this approach could be feasible to ameliorate muscle-wasting pathologies.

Novel cationic carotenoid lipids as delivery vectors of antisense oligonucleotides for exon skipping in Duchenne muscular dystrophy.

Duchenne Muscular Dystrophy (DMD) is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO)-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO) AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs). The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC).

Long-Term Systemic Treatment of a Mouse Model Displaying Chronic FSHD-like Pathology with Antisense Therapeutics That Inhibit DUX4 Expression.

Silencing the expression of the double homeobox 4 (DUX4) gene offers great potential for the treatment of facioscapulohumeral muscular dystrophy (FSHD). Several research groups have recently reported promising results using systemic antisense therapy in a transgenic small animal model of FSHD, the ACTA1-MCM/FLExDUX4 mouse model. However, the treatment was applied in non-DUX4-induced mice or shortly after DUX4 activation, which resulted in conditions that do not correctly represent the situation in a clinic. Here, we generated progressive FSHD-like pathology in ACTA1-MCM/FLExDUX4 mice and then treated the animals with vivoPMO-PACS4, an antisense compound that efficiently downregulates DUX4. To best mimic the translation of this treatment in clinical settings, the systemic antisense oligonucleotide administration was delayed to 3 weeks after the DUX4 activation so that the pathology was established at the time of the treatment. The chronic administration of vivoPMO-PACS4 for 8 weeks downregulated the DUX4 expression by 60%. Consequently, the treated mice showed an increase by 18% in body-wide muscle mass and 32% in muscle strength, and a reduction in both myofiber central nucleation and muscle fibrosis by up to 29% and 37%, respectively. Our results in a more suitable model of FSHD pathology confirm the efficacy of vivoPMO-PACS4 administration, and highlight the significant benefit provided by the long-term treatment of the disease.

Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment.

Duchenne muscular dystrophy (DMD) is a rare genetic disease affecting 1 in 5000 newborn boys. It is caused by mutations in the DMD gene with a consequent lack of dystrophin protein that leads to deterioration of myofibers and their replacement with fibro-adipogenic tissue. Using antisense oligonucleotides (AONs) to modify out-of-frame mutations in the DMD gene, named exon skipping, is currently considered among the most promising treatments for DMD patients. The development of this strategy is rapidly moving forward, and AONs designed to skip exons 51 and 53 have received accelerated approval in the USA. In preclinical setting, the mdx mouse model, carrying a point mutation in exon 23 of the murine Dmd gene that prevents production of dystrophin protein, has emerged as a valuable tool, and it is widely used to study in vivo therapeutic approaches for DMD. Here we describe the methodology for intravenous delivery of AONs targeting dystrophin through tail vein of mdx mice. Furthermore, the most relevant functional analyses to be performed in living mice, and the most informative histopathological and molecular assays to evaluate the effect of this treatment are detailed.

Optimized lentiviral vector to restore full-length dystrophin via a cell-mediated approach in a mouse model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a muscle wasting disorder caused by mutations in the DMD gene. Restoration of full-length dystrophin protein in skeletal muscle would have therapeutic benefit, but lentivirally mediated delivery of such a large gene in vivo has been hindered by lack of tissue specificity, limited transduction, and insufficient transgene expression. To address these problems, we developed a lentiviral vector, which contains a muscle-specific promoter and sequence-optimized full-length dystrophin, to constrain dystrophin expression to differentiated myotubes/myofibers and enhance the transgene expression. We further explored the efficiency of restoration of full-length dystrophin in vivo, by grafting DMD myoblasts that had been corrected by this optimized lentiviral vector intramuscularly into an immunodeficient DMD mouse model. We show that these lentivirally corrected DMD myoblasts effectively reconstituted full-length dystrophin expression in 93.58% ± 2.17% of the myotubes in vitro. Moreover, dystrophin was restored in 64.4% ± 2.87% of the donor-derived regenerated muscle fibers in vivo, which were able to recruit members of the dystrophin-glycoprotein complex at the sarcolemma. This study represents a significant advance over existing cell-mediated gene therapy strategies for DMD that aim to restore full-length dystrophin expression in skeletal muscle.