Search

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]