Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder disease which is characterized by progressive muscle degeneration or weakness due to the loss of functional dystrophin expression. For use as a cell-based disease model, we generated an induced pluripotent stem cell (iPSC) line (NCHi026-A) from fibroblasts derived from a skin biopsy of a 13-year-old patient with a nucleotide deletion across the DMD intron 54/exon55 junction (c.8028-501_8078del). The resulting cell line NCHi026-A was free of transgenes, expressed pluripotency-associated stem cell markers, maintained the normal karyotype and could be differentiated into three germ layers in vitro.

Duchenne muscular dystrophy (DMD) is caused by a wide variety of mutations that disrupt the reading frame of the DMD gene, leading to the absence of dystrophin. Several therapies have been explored, but, to date, there is no curative treatment for this disease. One reason for this is the lack of good models to test personalized therapy, as DMD is caused by more than 8558 different mutations. Although several DMD mouse models have been created, with some carrying the human dystrophin gene, it is practically impossible to generate a mouse model for each unique mutation identified in humans. Thus, patient-derived cell lines are the best option to study the impact of specific mutations and to screen potential therapies. While primary myoblasts derived from muscle biopsies are the most relevant model, they have limited proliferative capacity. To overcome this limitation, the immortalization of human primary myoblasts has been explored as an alternative. However, acquisition of the cells remains dependent on invasive muscle biopsies. In contrast, skin biopsies offer a less invasive and more accessible option. By immortalizing and transdifferentiating fibroblasts derived from skin biopsies into myoblasts, it is possible to establish a cell source with robust myogenic potential. This protocol describes a rapid and direct method for transdifferentiating fibroblasts into a myogenic lineage. The process involves transduction with two lentiviruses: one carrying hTERT for primary culture immortalization and another with a tetracycline-inducible MyoD. Upon the addition of doxycycline, MyoD expression induces the conversion of fibroblasts into myoblasts and subsequently mature myotubes expressing DMD mRNA and late differentiation markers, including dystrophin. This efficient transdifferentiation protocol serves as a valuable tool for investigating the effect of mutations in the DMD gene and exploring innovative gene-based or pharmacological biotherapies for DMD and other neuromuscular disorders.

The field of gene therapy for neuromuscular dystrophies has evolved over the past two decades. Despite some outstanding positive outcomes, some unfortunate adverse effects also led to big setbacks. One important key point is to study relevant preclinical models and to embrace diverse strategies to mitigate or avoid such negative outcomes. Although at first, for some diseases, the promise of a one-treatment-for-all approach was envisioned, it has recently become clear that a personalized approach will likely be preferable given the high variability in response between individuals.

Mouse models carrying specific subsets of mutations are particularly suitable for personalized medicine research. Nonetheless, mouse models do not completely mimic the human phenotype. This is the case for different Duchenne muscular dystrophy (DMD) mouse models, in which several aspects like muscle force, histopathology, cognition, and behavior can vary considerably, confounding the interpretation of therapy outcomes in preclinical studies. Two main common mutations in the DMD gene are exon 2 duplication and exon 45 deletion (1% and 4% of DMD patients respectively). Two mouse models carrying these mutations have been previously developed, named Dup2 and hDMDdel45, respectively. However, detailed studies on the locomotor, functional, behavioral, and histopathological aspects are lacking and would be beneficial for studies assessing treatment outcomes. This study also compares those features to the first mouse model identified for DMD, the mdx. We assessed histopathological features (fibrosis, inflammation, and centronucleation), isometric and isotonic (eccentric contraction) muscle force generation, locomotion (hang-wire and rotarod tests), and behavioral (open-field test) features in those mice. Our studies show that Dup2 and hDMDdel45 mice present significant deficits in function and behavior. Diaphragm and skeletal muscles have elevated levels of inflammation and fibrosis in comparison to wild-type controls. This study demonstrated that Dup2 and hDMDdel45 mice have the same degree of pathology as the classical DMD mouse model, the mdx.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease, caused by mutations in DMD gene leading to absence of dystrophin. 5-6% of DMD patients have mutations in exons 6-8 encoding for the second calponin homology domain of dystrophin. To test the efficiency of a novel vectorized exon skipping (VES) for patients with these mutations, we developed a multiple VES constructs that was screened in vitro in HEK293 and patient fibroblasts differentiated into myotubes to select our best VES candidates that mediate exon 6-8 skipping. In addition, we tested our lead candidates in a new humanized hDMD mdx mouse called hDMDm7 that was generated using CRISPR/Cas9. This mouse model does not express murine dystrophin and additionally contains a nonsense mutation in exon 7 (hDMDm7) resulting in no human dystrophin expression. Intramuscular (I.M.) injections using selected VES constructs were performed for hDMDm7 mice at 6 and 7 weeks of age. Exon skipping was evaluated by RT-PCR, dystrophin expression by WB and IF, and muscle force using electrophysiology. This mouse model presents a reduction of muscle integrity with centronucleation (>90% at 3 and 6 months of age) using H&E staining. Little to no dystrophin expression was determined by western blot, and the average percent drop over 10 consecutive eccentric contractions was 59% in hDMDm7 vs 9% in hDMD. Four lead VES constructs were selected and evaluated in the hDMDm7. One VES construct was tested effective in muscle force measurement at 3 months of post I.M. injection, which brought the percent drop of eccentric contraction back to 40%. This exon skipping strategy was able to restore muscle functions following I.M. treatment for hDMDm7 mouse model. This VES for exons 6-8 could be beneficial for 5-6% of DMD patients.

Exon skipping, a promising modality to treat Duchenne muscular dystrophy (DMD) is based on restoring the reading frame of the DMD pre-mRNA and to make truncated but functional dystrophin. Conventional exon skipping using antisense oligonucleotides has some limitations – a need of repeated injections due to less stability and limited tissue penetration in major affected tissues (e.g., heart). To overcome these, we and others used AAV.U7 antisense delivery. U7 small nuclear RNA carrying antisense sequence improves the stability and adeno-associated virus (AAV) increases tropism. This approach is currently being tested in a clinical trial for DMD exon 2 duplication. In this study, we applied this vectorized exon skipping strategy (VES) approach to a mutational hot spot in the DMD gene: the exon 44. We evaluated our lead candidate using intramuscular (IM) and systemic (IV) pre-clinical dose escalation study. Humanized DMD mice with exon 45 deletion were used. We evaluated exon skipping by RT-PCR, proteins restoration using immunostaining, muscle function using force transducer, muscle co-ordination and strength using rotarod and hang wire, and behavior test with open field apparatus. 3-months post-IM, around 85% of exon skipping was achieved, resulting in around 90% of truncated dystrophin expression and more than 50% improvement in eccentric contraction. 3-months post-IV dose escalation, around 80% of exon skipping was observed in heart and 30–60% in diaphragm, TA and gastrocnemius muscles. Dystrophin expression and improvement in force generation in dose-dependent manner was noted. Macrophage infiltration, muscle fibrosis, rotarod, hang wire and open field test revealed improvement after treatment with the highest IV dose. To conclude, our lead candidate induces efficient DMD exon 44 skipping, resulting in dystrophin production and muscle strength improvement in DMD. This AAV.U7-exon 44 skipping vector represents a promising candidate for ∼6-12% of DMD patients.

Patients with osteosarcoma (OS), a debilitating pediatric bone malignancy, have limited treatment options to combat aggressive disease. OS thrives on insulin growth factor (IGF)-mediated signaling that can facilitate cell proliferation. Previous efforts to target IGF-1R signaling were mostly unsuccessful, likely due to compensatory signaling through alternative splicing of the insulin receptor (IR) to the proliferative IR-A isoform. Here, we leverage splice-switching oligonucleotides (SSOs) to mitigate IR splicing toward the IR-B isoform. We show that SSOs can modulate cancer cell hallmarks and anoikis-resistant growth. Furthermore, we engineered the SSO sequence in an U7 snRNA packaged in an adeno-associated virus (AAV) to test the feasibility of viral vector-mediated gene therapy delivery. We noted modest increases in IR-B isoform levels after virus transduction, which prompted us to investigate the role of combinatorial treatments with dalotuzumab, an anti-IGF-1R monoclonal antibody. After observing additive impacts on phosphoprotein phosphorylation and anoikis-resistant growth with the dalotuzumab and SSO combination, we treated OS cells with dalotuzumab and the AAVrh74.U7 snRNA IR virus, which significantly slowed OS cell proliferation. While these viruses require further optimization, we highlight the potential for SSO therapy and viral vector delivery, as it may offer new treatment avenues for OS patients and be translated to other cancers.

Duchenne muscular dystrophy (DMD) is a severe form of muscular dystrophy affecting 1 in 5,200 males and characterized by progressive muscle weakness, leading to premature death due to cardiac and respiratory failure. Currently approved and experimental gene therapies have resulted in expression of multiple internally deleted, partially functional dystrophin isoforms, but overall, they demonstrate limited efficacy. An alternative strategy to induce exon skipping is to incorporate antisense oligonucleotides into U7 small nuclear RNA (snRNA), U7snRNA, and deliver them using adeno-associated virus (AAV). This approach has shown significant potential for restoring either full-length dystrophin or a highly functional N-terminal truncated protein in patients with exon 2 duplications (NCT04240314), which is the most frequent single exon duplication identified. Here, we tested the efficiency of rAAV.U7snRNA-mediated therapy to correct disrupted open reading frame (ORF) in DMD patients carrying skip-amenable mutations (duplications and deletions) that flank exon 17. In DMD patients with exon 17 duplications (Dup17), which is the second most common single exon duplication, the exclusion of a duplicated copy of exon 17 will result in WT DMD transcript and therefore expression of full-length dystrophin protein. In patients with variable deletions that flank exon 17, the exclusion of this exon will result in an In-frame DMD mRNA that encodes an internally deleted, yet highly functional dystrophin protein. In the case of small deletions (one or several exons), this will preserve most of the primary structure of the protein, retaining the biochemical function of dystrophin intact. We designed several AAV constructs, each encoding one copy of an antisense U7snRNA sequence targeting either splice acceptor (SAS), splice donor (SDS), or exon splicing enhancer (ESE) sites, and tested them in vitro and in vivo. Two DMD patient-derived cell lines—Immortalized tet-inducible-MyoD fibroblasts (FM cells)—carrying exon 17 amenable deletions were used to test our approach in vitro. Cells were treated with rAAV.U7snRNA vectors at four different doses and then differentiated into myotubes to study dystrophin expression. The effect of the treatment was evaluated using RT-PCR, which revealed robust exon 17 skipping in a dose-dependent manner in both cell lines tested. Semi-quantitative analysis of agarose gel images also confirmed the presence of In-frame dystrophin mRNA transcripts in both cell lines. To assess the efficiency of the vectors to induce exon 17 slipping in vivo, we generated a new DMD mouse model harboring an out-of-frame (OOF) deletion of exons 18-41 (Del18-41). All vectors were intramuscularly (IM) injected into the tibialis anterior (TA) and gastrocnemius (Gast) muscles of Del18-41 mice, which were euthanized 4 weeks post AAV administration. The efficiency of exon 17 skipping and dystrophin restoration was assessed by RT-PCR and JESS capillary western blotting (WB). RT-PCR analysis confirmed exon 17 exclusion for two of the three target sequences in both TA and Gast muscles, resulting in dystrophin protein levels up to 5.2% detected by JESS WB. These results clearly demonstrate that rAAV.U7snRNA-mediated therapy is a powerful therapy that can benefit up to 5.5% of all DMD patients carrying amenable mutations flanking exon 17.

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease, caused by mutations in DMD gene leading to absence of dystrophin. Additionally, around one-third of patients present cognitive impairments. Several therapeutic approaches including exon-skipping have used efficient systemic delivery of adeno-associated virus (AAV). However, the high dose of vector needed to treat adult patients represents a risk, especially for the liver. We have investigated an alternative administration route using cerebrospinal fluid (CSF) delivery to avoid potential liver toxicity and improve brain transduction. Newborn C57BL/6 mice were intracerebroventricular (ICV) or face vein (FV) injected with 1E+11vg scAAV9.CBA.GFP to study virus biodistribution. After one and three months (mo) post-injection (pi), similar levels of GFP expression were observed in all muscles for CSF and systemic groups. However, a higher expression was detected in the brain after ICV injection and liver was less transduced by ICV injection compared to FV, which was confirmed by RT-PCR and WB. To conclude, CSF and systemic delivery allowed similar levels of muscle transduction by AAV9, although the brain transduction after ICV injection was superior and liver was less transduced, supporting the use of CSF delivery for the simultaneous distribution of AAV9 to muscles and CNS.

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults affecting the skeletal muscle, heart, and brain. It is caused by a CTG repeat expansion in the 3’UTR of the DMPK gene. The CUG repeats aggregates with muscle-blind proteins (MBNLs) in nuclear foci, leading to the impairment of several mRNA processing functions, including alternative splicing. The reversal from an adult to a fetal splicing profile alters or abrogates the function of a myriad of proteins, resulting in dysfunction in multiple organs. Currently, there is no treatment available for DM1 patients. Antisense oligonucleotides (ASOs) aiming to knock down DMPK expression or bind to the CUG repeats and release MBNL have been explored, with promising results in animal models. Unfortunately, a clinical trial with these drugs, while safe, failed to show improvement in patients, probably due to intrinsic limitations of ASOs. To overcome this limitation, we designed modified U7 small nuclear RNAs containing a promoter and antisense sequences targeting the 3’UTR region to promote DMPK knockdown or steric hindrance of the CUG repeats. We used patient-derived cell lines to test our approach, and the effect of the treatment was evaluated using RNA FISH combined with MBNL1 immunostaining for foci quantification and MBNL1 localization. Gene expression and splicing profiles were assessed by RNAseq and selected candidate genes were confirmed by RT-PCR. The AAV treatment reduced the foci number, released MBNL1 from the CUG foci, and shifted the splicing profile. Based on the in vitro results, we selected one vector candidate to test in vivo. We injected AAV1.U7snRNAs in the tibialis anterior (TA) and gastrocnemius (Gas) of HSAlr mice, which express CUG repeats in skeletal muscles only. Eight weeks post-injection, we submitted the animals to muscle torque and electromyography (EMG) tests and collected the muscles for further analysis. The functional tests revealed reduced mechanical myotonia (a major phenotype in DM1 muscles) and even the absence of electrical myotonia in individual muscles. RNA FISH quantification showed a reduced number and intensity of foci in TA and Gas. The reduction in foci led to improved splicing of Serca1, Mbnl1, Ldb3, and Clcn1 transcripts. In conclusion, U7snRNAs that interfere with the CUG-expanded mRNA can reverse DM1 pathology with the restoration of molecular features in vitro and in vivo, accompanied by significant amelioration of muscle physiological properties. This work will provide the basis for testing the first AAV-based gene therapy for DM1 in humans.

Schwann cells (SCs) have a critical role in the peripheral nervous system. These cells are able to support axons during homeostasis and after injury. However, mutations in genes associated with the SCs repair program or myelination result in dysfunctional SCs. Several neuropathies such as Charcot–Marie–Tooth (CMT) disease, diabetic neuropathy and Guillain–Barré syndrome show abnormal SC functions and an impaired regeneration process. Thus, understanding SCs-axon interaction and the nerve environment in the context of homeostasis as well as post-injury and disease onset is necessary. Several neurotrophic factors, cytokines, and regulators of signaling pathways associated with proliferation, survival and regeneration are involved in this process. Preclinical studies have focused on the discovery of therapeutic targets for peripheral neuropathies and injuries. To study the effect of new therapeutic targets, modeling neuropathies and peripheral nerve injuries (PNIs) in vitro and in vivo are useful tools. Furthermore, several in vitro protocols have been designed using SCs and neuron cell lines to evaluate these targets in the regeneration process. SCs lines have been used to generate effective myelinating SCs without success. Alternative options have been investigated using direct conversion from somatic cells to SCs or SCs derived from pluripotent stem cells to generate functional SCs. This review will go over the advantages of these systems and the problems associated with them. In addition, there have been challenges in establishing adequate and reproducible protocols in vitro to recapitulate repair SC-neuron interactions observed in vivo. So, we also discuss the mechanisms of repair SCs-axon interactions in the context of peripheral neuropathies and nerve injury (PNI) in vitro and in vivo. Finally, we summarize current preclinical studies evaluating transgenes, drug, and novel compounds with translational potential into clinical studies.