Novel MECP2 gene therapy is effective in a multicenter study using two mouse models of Rett syndrome and is safe in non-human primates.

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.

Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy.

DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated from 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Reverse transcription-polymerase chain reaction or high-throughput RNA sequencing methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3'-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.

Clinical phenotypes as predictors of the outcome of skipping around DMD exon 45.

OBJECTIVE: Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion-exon 45 (Δ45)-may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. METHODS: Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy were analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. RESULTS: As expected, deletions of either exons 45 to 47 (Δ45-47) or exons 45 to 48 (Δ45-48) result in BMD in 97% (36 of 37) of subjects. Unexpectedly, deletion of exons 45 to 46 (Δ45-46) is associated with the more severe DMD phenotype in 4 of 4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44 to 45 (Δ44-45) were found within the United Dystrophinopathy Project database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. INTERPRETATION: The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of exon 44 and multiexon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone.

The ZZ domain of dystrophin in DMD: making sense of missense mutations.

Duchenne muscular dystrophy (DMD) is associated with the loss of dystrophin, which plays an important role in myofiber integrity via interactions with ß-dystroglycan and other members of the transmembrane dystrophin-associated protein complex. The ZZ domain, a cysteine-rich zinc-finger domain near the dystrophin C-terminus, is implicated in forming a stable interaction between dystrophin and ß-dystroglycan, but the mechanism of pathogenesis of ZZ missense mutations has remained unclear because not all such mutations have been shown to alter ß-dystroglycan binding in previous experimental systems. We engineered three ZZ mutations (p.Cys3313Phe, p.Asp3335His, and p.Cys3340Tyr) into a short construct similar to the Dp71 dystrophin isoform for in vitro and in vivo studies and delineated their effect on protein expression, folding properties, and binding partners. Our results demonstrate two distinct pathogenic mechanisms for ZZ missense mutations. The cysteine mutations result in diminished or absent subsarcolemmal expression because of protein instability, likely due to misfolding. In contrast, the aspartic acid mutation disrupts binding with ß-dystroglycan despite an almost normal expression at the membrane, confirming a role for the ZZ domain in ß-dystroglycan binding but surprisingly demonstrating that such binding is not required for subsarcolemmal localization of dystrophin, even in the absence of actin binding domains.

Neuron-Schwann cell interactions in peripheral nervous system homeostasis, disease, and preclinical treatment.

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.

Persistence of exon 2 skipping and dystrophin expression at 18 months after U7snRNA-mediated therapy in the Dup2 mouse model.

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.

Promising AAV.U7snRNAs vectors targeting DMPK improve DM1 hallmarks in patient-derived cell lines.

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.

UMD-DYSF, a novel locus specific database for the compilation and interactive analysis of mutations in the dysferlin gene.

Mutations in the dysferlin gene (DYSF) lead to a complete or partial absence of the dysferlin protein in skeletal muscles and are at the origin of dysferlinopathies, a heterogeneous group of rare autosomal recessive inherited neuromuscular disorders. As a step towards a better understanding of the DYSF mutational spectrum, and towards possible inclusion of patients in future therapeutic clinical trials, we set up the Universal Mutation Database for Dysferlin (UMD-DYSF), a Locus-Specific Database developed with the UMD® software. The main objective of UMD-DYSF is to provide an updated compilation of mutational data and relevant interactive tools for the analysis of DYSF sequence variants, for diagnostic and research purposes. In particular, specific algorithms can facilitate the interpretation of newly identified intronic, missense- or isosemantic-exonic sequence variants, a problem encountered recurrently during genetic diagnosis in dysferlinopathies. UMD-DYSF v1.0 is freely accessible at www.umd.be/DYSF/. It contains a total of 742 mutational entries corresponding to 266 different disease-causing mutations identified in 558 patients worldwide diagnosed with dysferlinopathy. This article presents for the first time a comprehensive analysis of the dysferlin mutational spectrum based on all compiled DYSF disease-causing mutations reported in the literature to date, and using the main bioinformatics tools offered in UMD-DYSF.

Emerging Perspectives on Gene Therapy Delivery for Neurodegenerative and Neuromuscular Disorders.

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.

Systemic delivery of an AAV9 exon-skipping vector significantly improves or prevents features of Duchenne muscular dystrophy in the Dup2 mouse.

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.

Systemic PPMO-mediated dystrophin expression in the Dup2 mouse model of Duchenne muscular dystrophy.

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.

Mechanisms of IRF2BPL-related disorders and identification of a potential therapeutic strategy.

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.

Translational research and therapeutic perspectives in dysferlinopathies.

Dysferlinopathies are autosomal recessive disorders caused by mutations in the dysferlin (DYSF) gene, encoding the dysferlin protein. DYSF mutations lead to a wide range of muscular phenotypes, with the most prominent being Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B) and the second most common being LGMD. Symptoms generally appear at the end of childhood and, although disease progression is typically slow, walking impairments eventually result. Dysferlin is a modular type II transmembrane protein for which numerous binding partners have been identified. Although dysferlin function is only partially elucidated, this large protein contains seven calcium sensor C2 domains, shown to play a key role in muscle membrane repair. On the basis of this major function, along with detailed clinical observations, it has been possible to design various therapeutic approaches for dysferlin-deficient patients. Among them, exon-skipping and minigene transfer strategies have been evaluated at the preclinical level and, to date, represent promising approaches for clinical trials. This review aims to summarize the pathophysiology of dysferlinopathies and to evaluate the therapeutic potential for treatments currently under development.

U7 snRNA, a Small RNA with a Big Impact in Gene Therapy.

The uridine-rich 7 (U7) small nuclear RNA (snRNA) is a component of a small nuclear ribonucleoprotein (snRNP) complex. U7 snRNA naturally contains an antisense sequence that identifies histone premessenger RNAs (pre-mRNAs) and is involved in their 3' end processing. By altering this antisense sequence, researchers have turned U7 snRNA into a versatile tool for targeting pre-mRNAs and modifying splicing. Encapsulating a modified U7 snRNA into a viral vector such as adeno-associated virus (also referred as vectorized exon skipping/inclusion, or VES/VEI) enables the delivery of this highly efficacious splicing modulator into a range of cell lines, primary cells, and tissues. In addition, and in contrast to antisense oligonucleotides, viral delivery of U7 snRNA enables long-term expression of antisense sequences in the nucleus as part of a stable snRNP complex. As a result, VES/VEI has emerged as a promising therapeutic platform for treating a large variety of human diseases caused by errors in pre-mRNA splicing or its regulation. Here we provide an overview of U7 snRNA's natural function and its applications in gene therapy.

Designed U7 snRNAs inhibit DUX4 expression and improve FSHD-associated outcomes in DUX4 overexpressing cells and FSHD patient myotubes.

Facioscapulohumeral muscular dystrophy (FSHD) arises from epigenetic changes that de-repress the DUX4 gene in muscle. The full-length DUX4 protein causes cell death and muscle toxicity, and therefore we hypothesize that FSHD therapies should center on inhibiting full-length DUX4 expression. In this study, we developed a strategy to accomplish DUX4 inhibition using U7-small nuclear RNA (snRNA) antisense expression cassettes (called U7-asDUX4). These non-coding RNAs were designed to inhibit production or maturation of the full-length DUX4 pre-mRNA by masking the DUX4 start codon, splice sites, or polyadenylation signal. In so doing, U7-asDUX4 snRNAs operate similarly to antisense oligonucleotides. However, in contrast to oligonucleotides, which are limited by poor uptake in muscle and a requirement for lifelong repeated dosing, U7-asDUX4 snRNAs can be packaged within myotropic gene therapy vectors and may require only a single administration when delivered to post-mitotic cells in vivo. We tested several U7-asDUX4s that reduced DUX4 expression in vitro and improved DUX4-associated outcomes. Inhibition of DUX4 expression via U7-snRNAs could be a new prospective gene therapy approach for FSHD or be used in combination with other strategies, like RNAi therapy, to maximize DUX4 silencing in individuals with FSHD.

Direct Reprogramming of Human Fibroblasts into Myoblasts to Investigate Therapies for Neuromuscular Disorders.

Investigations into both the pathophysiology and therapeutic targets in muscular dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Several mouse models have been created but they either do not truly represent the human physiopathology of the disease or are not representative of the broad spectrum of mutations found in humans. The immortalization of human primary myoblasts is an alternative to this limitation; however, it is still dependent on muscle biopsies, which are invasive and not easily available. In contrast, skin biopsies are easier to obtain and less invasive to patients. Fibroblasts derived from skin biopsies can be immortalized and transdifferentiated into myoblasts, providing a source of cells with excellent myogenic potential. Here, we describe a fast and direct reprogramming method of fibroblast into a myogenic lineage. Fibroblasts are transduced with two lentiviruses: hTERT to immortalize the primary culture and a tet-inducible MYOD, which upon the addition of doxycycline, induces the conversion of fibroblasts into myoblasts and then mature myotubes, which express late differentiation markers. This quick transdifferentiation protocol represents a powerful tool to investigate pathological mechanisms and to investigate innovative gene-based or pharmacological biotherapies for neuromuscular disorders.

Absence of Significant Off-Target Splicing Variation with a U7snRNA Vector Targeting DMD Exon 2 Duplications.

Exon skipping therapies for Duchenne muscular dystrophy that restore an open reading frame can be induced by the use of noncoding U7 small nuclear RNA (U7snRNA) modified by an antisense exon-targeting sequence delivered by an adeno-associated virus (AAV) vector. We have developed an AAV vector (AAV9.U7-ACCA) containing four U7snRNAs targeting the splice donor and acceptor sites of dystrophin exon 2, resulting in highly efficient exclusion of DMD exon 2. We assessed the specificity of splice variation induced by AAV9.U7-ACCA delivery in the Dmd exon 2 duplication (Dup2) mouse model through an unbiased RNA-seq approach. Treatment-related effects on pre-mRNA splicing were quantified using local splicing variation (LSV) analysis. Filtering the transcriptome for differences in treatment-related splicing resulted in only 16 candidate off-target LSVs. Only a single candidate off-target LSV was found in both skeletal and cardiac muscle tissue and occurred at a known variable cassette exon. In contrast, four LSVs represented significant on-target correction of Dmd exon 2 splicing and transcriptome analysis showed correction of known dystrophin-deficient gene dysregulation. We conclude that the absence of off-target splicing induced by treatment with the U7-ACCA vector supports the continued clinical development of this approach.

Pre-clinical dose-escalation studies establish a therapeutic range for U7snRNA-mediated DMD exon 2 skipping.

Duchenne muscular dystrophy (DMD) is an X-linked progressive disease characterized by loss of dystrophin protein that typically results from truncating mutations in the DMD gene. Current exon-skipping therapies have sought to treat deletion mutations that abolish an open reading frame (ORF) by skipping an adjacent exon, in order to restore an ORF that allows translation of an internally deleted yet partially functional protein, as is seen with many patients with the milder Becker muscular dystrophy (BMD) phenotype. In contrast to that approach, skipping of one copy of a duplicated exon would be expected to result in a full-length transcript and production of a wild-type protein. We have developed an adeno-associated virus (AAV)-based U7snRNA exon-skipping approach directed toward exon 2, duplications of which represent 10% of all DMD duplication mutations. Deletion of exon 2 results in utilization of an exon 5 internal ribosome entry site (IRES) that allows translation beginning in exon 6 of a highly protective dystrophin protein, providing a wide therapeutic window for treatment. Both intramuscular and systemic administration of this vector in the Dup2 mouse model results in robust dystrophin expression and correction of muscle physiologic defects, allowing dose escalation to establish a putative minimal efficacious dose for a human clinical trial.

Lack of Toxicity in Nonhuman Primates Receiving Clinically Relevant Doses of an AAV9.U7snRNA Vector Designed to Induce DMD Exon 2 Skipping.

Therapeutic exon skipping as a treatment for Duchenne muscular dystrophy (DMD) has largely concentrated on the delivery of antisense oligomers to treat out-of-frame exon deletions. Here we report on the preclinical development of an adeno-associated virus (AAV)-encapsidated viral vector containing four copies of the noncoding U7 small nuclear RNA (U7snRNA), each targeted to either the splice donor or the splice acceptor sites of DMD exon 2. We have previously shown that delivery of this vector (scAAV9.U7.ACCA) to the Dup2 mouse model results in expression of full-length dystrophin from wild-type DMD mRNA, as well as an internal ribosome entry site (IRES)-driven isoform translated only in the absence of exon 2 (deletion exon 2 [Del2] mRNA). Here we present the data from a rigorous dose escalation toxicity study in nonhuman primates, encompassing two doses (3 × 1013 and 8 × 1013 vg/kg) and two time points (3 and 6 months postinjection). No evidence for significant toxicity was seen by biochemical, histopathologic, or clinical measures, providing evidence for safety that led to initiation of a first-in-human clinical trial.

Efficient bypass of mutations in dysferlin deficient patient cells by antisense-induced exon skipping.

Mutations in DYSF encoding dysferlin cause primary dysferlinopathies, autosomal recessive diseases that mainly present clinically as Limb Girdle Muscular Dystrophy type 2B and Miyoshi myopathy. More than 350 different sequence variants have been reported in DYSF. Like dystrophin, the size of the dysferlin mRNA is above the limited packaging size of AAV vectors. Alternative strategies to AAV gene transfer in muscle cells must then be addressed for patients. A gene therapy approach for Duchenne muscular dystrophy was recently developed, based on exon-skipping strategy. Numerous sequences are recognized by splicing protein complexes and, when specifically blocked by antisense oligoucleotides (AON), the corresponding exon is skipped. We hypothesized that this approach could be useful for patients affected with dysferlinopathies. To confirm this assumption, exon 32 was selected as a prioritary target for exon skipping strategy. This option was initially driven by the report from Sinnreich and colleagues of a patient with a very mild and late-onset phenotype associated to a natural skipping of exon 32. Three different antisense oligonucleotides were tested in myoblasts generated from control and patient MyoD transduced fibroblasts, either as oligonucleotides or after incorporation into lentiviral vectors. These approaches led to a high efficiency of exon 32 skipping. Therefore, these results seem promising, and could be applied to several other exons in the DYSF gene. Patients carrying mutations in exons whose the in-frame suppression has been proven to have no major consequences on the protein function, might benefit of exon-skipping based gene correction.