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-mediated exon skipping is a promising approach for the treatment of Duchenne muscular dystrophy (DMD), a rare life-threatening genetic disease due to dystrophin deficiency. Such an approach can restore the disrupted reading frame of dystrophin pre-mRNA, generating a truncated form of the protein. Alternatively, antisense therapy can be used to induce destructive exon skipping of myostatin pre-mRNA, knocking down myostatin expression to enhance muscle strength and reduce fibrosis. We have reported previously that intramuscular or intraperitoneal antisense administration inducing dual exon skipping of dystrophin and myostatin pre-mRNAs was beneficial in mdx mice, a mouse model of DMD, although therapeutic effects were muscle type restricted, possibly due to the delivery routes used. Here, following systemic intravascular antisense treatment, muscle strength and body activity of treated adult mdx mice increased to the levels of healthy controls. Importantly, hallmarks of muscular dystrophy were greatly improved in mice receiving the combined exon-skipping therapy, as compared to those receiving dystrophin antisense therapy alone. Our results support the translation of antisense therapy for dystrophin restoration and myostatin inhibition into the clinical setting for DMD.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.

Fibrosis results from the excessive accumulation of extracellular matrix in chronically injured tissue. The fibrotic process is governed by crosstalk between many signaling pathways. The search for an effective treatment is further complicated by the fact that there is a degree of tissue-specificity in the pathways involved, although the process is not completely understood for all tissues. A plethora of drugs have shown promise in pre-clinical models, which is not always borne out translationally in clinical trial. With the recent approvals of two antisense oligonucleotides for the treatment of the genetic diseases Duchenne muscular dystrophy and spinal muscular atrophy, we explore here the potential of antisense oligonucleotides to knockdown the expression of pro-fibrotic proteins. We give an overview of the generalized fibrotic process, concentrating on key players and highlight where antisense oligonucleotides have been used effectively in cellular and animal models of different fibrotic conditions. Consideration is given to the advantages antisense oligonucleotides would have as an anti-fibrotic therapy alongside factors that would need to be addressed to improve efficacy. A prospective outlook for the development of antisense oligonucleotides to target fibrosis is outlined.

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.

Standard integration-proficient lentiviral vectors (IPLVs) are effective at much lower doses than other vector systems and have shown promise for gene therapy of Parkinson's disease (PD). Their main drawback is the risk of insertional mutagenesis. The novel biosafety-enhanced integration-deficient lentiviral vectors (IDLVs) may offer a significant enhancement in biosafety, but have not been previously tested in a model of a major disease. We have assessed biosafety and transduction efficiency of IDLVs in a rat model of PD, using IPLVs as a reference. Genomic insertion of lentivectors injected into the lesioned striatum was studied by linear amplification-mediated polymerase chain reaction (PCR), followed by deep sequencing and insertion site analysis, demonstrating lack of significant IDLV integration. Reporter gene expression studies showed efficient, long-lived, and transcriptionally targeted expression from IDLVs injected ahead of lesioning in the rat striatum, although at somewhat lower expression levels than from IPLVs. Transgenic human glial cell line-derived neurotrophic factor ( hGDNF ) expression from IDLVs was used for a long-term investigation of lentivector-mediated, transcriptionally targeted neuroprotection in this PD rat model. Vectors were injected before striatal lesioning, and the results showed improvements in nigral dopaminergic neuron survival and behavioral tests regardless of lentiviral integration proficiency, although they confirmed lower expression levels of hGDNF from IDLVs. These data demonstrate the effectiveness of IDLVs in a model of a major disease and indicate that these vectors could provide long-term PD treatment at low dose, combining efficacy and biosafety for targeted central nervous system applications.