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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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., Competing Interests: The AAVrh74.U7 snRNA viruses shown in this work are the subject of an international patent application (USSN 63/572,178) with A.S.V., P.Y.W., N.W., D.S.C., and T.P.C. as inventors. T.P.C. is a member of the Molecular Therapy Oncology board and Editor-in-Chief. D.S.C. is a member of the Molecular Therapy Oncology editorial board. F.R. is affiliated with Ionis Pharmaceuticals., (© 2024 The Authors.)

Duchenne muscular dystrophy (DMD) is a progressive X-linked disease caused by mutations in the DMD gene that prevent the expression of a functional dystrophin protein. Exon duplications represent 6%-11% of mutations, and duplications of exon 2 (Dup2) are the most common (∼11%) of duplication mutations. An exon-skipping strategy for Dup2 mutations presents a large therapeutic window. Skipping one exon copy results in full-length dystrophin expression, whereas skipping of both copies (Del2) activates an internal ribosomal entry site (IRES) in exon 5, inducing the expression of a highly functional truncated dystrophin isoform. We have previously confirmed the therapeutic efficacy of AAV9.U7snRNA-mediated skipping in the Dup2 mouse model and showed the absence of off-target splicing effects and lack of toxicity in mice and nonhuman primates. Here, we report long-term dystrophin expression data following the treatment of 3-month-old Dup2 mice with the scAAV9.U7.ACCA vector. Significant exon 2 skipping and robust dystrophin expression in the muscles and hearts of treated mice persist at 18 months after treatment, along with the partial rescue of muscle function. These data extend our previous findings and show that scAAV9.U7.ACCA provides long-term protection by restoring the disrupted dystrophin reading frame in the context of exon 2 duplications., Competing Interests: NCH licensed the vector described herein to Audentes Therapeutics, which was subsequently acquired by Astellas Therapeutics. N.W. and K.M.F. have received royalty payments as a result of this license., (© 2023 The Authors.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oliveira, Yanick, Wein and Gomez Limia.)

The AAV9 gene therapy vector presented in this study is safe in mice and non-human primates and highly efficacious without causing overexpression toxicity, a major challenge for clinical translation of Rett syndrome gene therapy vectors to date. Our team designed a new truncated methyl-CpG-binding protein 2 (MECP2) promoter allowing widespread expression of MECP2 in mice and non-human primates after a single injection into the cerebrospinal fluid without causing overexpression symptoms up to 18 months after injection. Additionally, this new vector is highly efficacious at lower doses compared with previous constructs as demonstrated in extensive efficacy studies performed by two independent laboratories in two different Rett syndrome mouse models carrying either a knockout or one of the most frequent human mutations of Mecp2. Overall, data from this multicenter study highlight the efficacy and safety of this gene therapy construct, making it a promising candidate for first-in-human studies to treat Rett syndrome., Competing Interests: Declaration of interests B.K.K. and NCH hold patent filings on this work and received royalties. B.B. received compensation for histopathological analysis., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults and affects mainly the skeletal muscle, heart, and brain. DM1 is caused by a CTG repeat expansion in the 3'UTR region of the DMPK gene that sequesters muscleblind-like proteins, blocking their splicing activity and forming nuclear RNA foci . Consequently, many genes have their splicing reversed to a fetal pattern. There is no treatment for DM1, but several approaches have been explored, including antisense oligonucleotides (ASOs) aiming to knock down DMPK expression or bind to the CTGs expansion. ASOs were shown to reduce RNA foci and restore the splicing pattern. However, ASOs have several limitations and although being safe treated DM1 patients did not demonstrate improvement in a human clinical trial. AAV-based gene therapies have the potential to overcome such limitations, providing longer and more stable expression of antisense sequences. In the present study, we designed different antisense sequences targeting exons 5 or 8 of DMPK and the CTG repeat tract aiming to knock down DMPK expression or promote steric hindrance, respectively. The antisense sequences were inserted in U7snRNAs, which were then vectorized in AAV8 particles. Patient-derived myoblasts treated with AAV8. U7snRNAs showed a significant reduction in the number of RNA foci and re-localization of muscle-blind protein. RNA-seq analysis revealed a global splicing correction in different patient-cell lines, without alteration in DMPK expression., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital licensed the vector described herein to Audentes/Astellas Therapeutics. NW and KF have received royalty payments as a result of this license. NW is a Scientific Advisor for Alcyone Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Almeida, Robriquet, Vetter, Huang, Neinast, Hernandez-Rosario, Rajakumar, Arnold, McBride, Flanigan, Weiss and Wein.)

The recently discovered neurological disorder NEDAMSS is caused by heterozygous truncations in the transcriptional regulator IRF2BPL. Here, we reprogram patient skin fibroblasts to astrocytes and neurons to study mechanisms of this newly described disease. While full-length IRF2BPL primarily localizes to the nucleus, truncated patient variants sequester the wild-type protein to the cytoplasm and cause aggregation. Moreover, patient astrocytes fail to support neuronal survival in coculture and exhibit aberrant mitochondria and respiratory dysfunction. Treatment with the small molecule copper ATSM (CuATSM) rescues neuronal survival and restores mitochondrial function. Importantly, the in vitro findings are recapitulated in vivo, where co-expression of full-length and truncated IRF2BPL in Drosophila results in cytoplasmic accumulation of full-length IRF2BPL. Moreover, flies harboring heterozygous truncations of the IRF2BPL ortholog (Pits) display progressive motor defects that are ameliorated by CuATSM treatment. Our findings provide insights into mechanisms involved in NEDAMSS and reveal a promising treatment for this severe disorder., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Neurodegenerative disorders (NDDs), such as Alzheimer's disease (AD) and Parkinson's Disease (PD), are a group of heterogeneous diseases that mainly affect central nervous system (CNS) functions. A subset of NDDs exhibit CNS dysfunction and muscle degeneration, as observed in Gangliosidosis 1 (GM1) and late stages of PD. Neuromuscular disorders (NMDs) are a group of diseases in which patients show primary progressive muscle weaknesses, including Duchenne Muscular Dystrophy (DMD), Pompe disease, and Spinal Muscular Atrophy (SMA). NDDs and NMDs typically have a genetic component, which affects the physiological functioning of critical cellular processes, leading to pathogenesis. Currently, there is no cure or efficient treatment for most of these diseases. More than 200 clinical trials have been completed or are currently underway in order to establish safety, tolerability, and efficacy of promising gene therapy approaches. Thus, gene therapy-based therapeutics, including viral or non-viral delivery, are very appealing for the treatment of NDDs and NMDs. In particular, adeno-associated viral vectors (AAV) are an attractive option for gene therapy for NDDs and NMDs. However, limitations have been identified after systemic delivery, including the suboptimal capacity of these therapies to traverse the blood-brain barrier (BBB), degradation of the particles during the delivery, high reactivity of the patient's immune system during the treatment, and the potential need for redosing. To circumvent these limitations, several preclinical and clinical studies have suggested intrathecal (IT) delivery to target the CNS and peripheral organs via cerebrospinal fluid (CSF). CSF administration can vastly improve the delivery of small molecules and drugs to the brain and spinal cord as compared to systemic delivery. Here, we review AAV biology and vector design elements, different therapeutic routes of administration, and highlight CSF delivery as an attractive route of administration. We discuss the different aspects of neuromuscular and neurodegenerative diseases, such as pathogenesis, the landscape of mutations, and the biological processes associated with the disease. We also describe the hallmarks of NDDs and NMDs as well as discuss current therapeutic approaches and clinical progress in viral and non-viral gene therapy and enzyme replacement strategies for those diseases.

Duchenne muscular dystrophy (DMD) is a devastating muscle-wasting disease that arises due to the loss of dystrophin expression, leading to progressive loss of motor and cardiorespiratory function. Four exon-skipping approaches using antisense phosphorodiamidate morpholino oligomers (PMOs) have been approved by the FDA to restore a DMD open reading frame, resulting in expression of a functional but internally deleted dystrophin protein, but in patients with single-exon duplications, exon skipping has the potential to restore full-length dystrophin expression. Cell-penetrating peptide-conjugated PMOs (PPMOs) have demonstrated enhanced cellular uptake and more efficient dystrophin restoration than unconjugated PMOs. In the present study, we demonstrate widespread PPMO-mediated dystrophin restoration in the Dup2 mouse model of exon 2 duplication, representing the most common single-exon duplication among patients with DMD. In this proof-of-concept study, a single intravenous injection of PPMO targeting the exon 2 splice acceptor site induced 45% to 68% exon 2-skipped Dmd transcripts in Dup2 skeletal muscles 15 days post-injection. Muscle dystrophin restoration peaked at 77% to 87% average dystrophin-positive fibers and 41% to 51% of normal signal intensity by immunofluorescence, and 15.7% to 56.8% of normal by western blotting 15 to 30 days after treatment. These findings indicate that PPMO-mediated exon skipping is a promising therapeutic strategy for muscle dystrophin restoration in the context of exon 2 duplications., Competing Interests: K.M.F. has received research support for a clinical trial from Sarepta Therapeutics. K.M.F. and N.W. hold a patent related to therapeutic exon 2 skipping, and along with Nationwide Children’s Hospital receive royalties from Astellas Gene Therapy for that patent, which is not directly related to the data presented here., (© 2022 The Authors.)

Duchenne muscular dystrophy (DMD) is typically caused by mutations that disrupt the DMD reading frame, but nonsense mutations in the 5' part of the gene induce utilization of an internal ribosomal entry site (IRES) in exon 5, driving expression of a highly functional N-truncated dystrophin. We have developed an AAV9 vector expressing U7 small nuclear RNAs targeting DMD exon 2 and have tested it in a mouse containing a duplication of exon 2, in which skipping of both exon 2 copies induces IRES-driven expression, and skipping of one copy leads to wild-type dystrophin expression. One-time intravascular injection either at postnatal days 0-1 or at 2 months results in efficient exon skipping and dystrophin expression, and significant protection from functional and pathologic deficits. Immunofluorescence quantification showed 33%-53% average dystrophin intensity and 55%-79% average dystrophin-positive fibers in mice treated in adulthood, with partial amelioration of DMD pathology and correction of DMD-associated alterations in gene expression. In mice treated neonatally, dystrophin immunofluorescence reached 49%-85% of normal intensity and 76%-99% dystrophin-positive fibers, with near-complete correction of dystrophic pathology, and these beneficial effects persisted for at least 6 months. Our results demonstrate the robustness, durability, and safety of exon 2 skipping using scAAV9.U7snRNA.ACCA, supporting its clinical use., Competing Interests: Since the performance of this work, Nationwide Children’s Hospital licensed the vector described herein to Audentes/Astellas Therapeutics. N.W., A.V., and K.M.F. have received royalty payments as a result of this license., (© 2022 The Authors.)