Dystrophin Restoration Research Articles

Dystrophin S3059 phosphorylation partially attenuates denervation atrophy in mouse tibialis anterior muscles.

The dystrophin protein has well-characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine-rich region of dystrophin enhances binding between dystrophin and β-dystroglycan, and mimicking phosphorylation at this site by site-directed mutagenesis attenuates myotube atrophy invitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting invivo, CRISPR-Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of β-dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation-induced atrophy, but may have more significant impact in less severe modes of muscle wasting.

Deletion of miR-146a enhances therapeutic protein restoration in model of dystrophin exon skipping

Duchenne muscular dystrophy (DMD) is a progressive muscle disease caused by the absence of dystrophin protein. One current DMD therapeutic strategy, exon skipping, produces a truncated dystrophin isoform using phosphorodiamidate morpholino oligomers (PMOs). However, the potential of exon skipping therapeutics has not been fully realized as increases in dystrophin protein have been minimal in clinical trials. Here, we investigate how miR-146a-5p, which is highly elevated in dystrophic muscle, impacts dystrophin protein levels. We find inflammation strongly induces miR-146a in dystrophic, but not wild-type myotubes. Bioinformatics analysis reveals that the dystrophin 3′ UTR harbors a miR-146a binding site, and subsequent luciferase assays demonstrate miR-146a binding inhibits dystrophin translation. In dystrophin-null mdx52 mice, co-injection of miR-146a reduces dystrophin restoration by an exon 51 skipping PMO. To directly investigate how miR-146a impacts therapeutic dystrophin rescue, we generated mdx52 with body-wide miR-146a deletion (146aX). Administration of an exon skipping PMO via intramuscular or intravenous injection markedly increases dystrophin protein levels in 146aX versus mdx52 muscles; skipped dystrophin transcript levels are unchanged, suggesting a post-transcriptional mechanism-of-action. Together, these data show that miR-146a expression opposes therapeutic dystrophin restoration, suggesting miR-146a inhibition warrants further research as a potential DMD exon skipping co-therapy.

Generalizable anchor aptamer strategy for loading nucleic acid therapeutics on exosomes.

Clinical deployment of oligonucleotides requires delivery technologies that improve stability, target tissue accumulation and cellular internalization. Exosomes show potential as ideal delivery vehicles. However, an affordable generalizable system for efficient loading of oligonucleotides on exosomes remain lacking. Here, we identified an Exosomal Anchor DNA Aptamer (EAA) via SELEX against exosomes immobilized with our proprietary CP05 peptides. EAA shows high binding affinity to different exosomes and enables efficient loading of nucleic acid drugs on exosomes. Serum stability of thrombin inhibitor NU172 was prolonged by exosome-loading, resulting in increased blood flow after injury in vivo. Importantly, Duchenne Muscular Dystrophy PMO can be readily loaded on exosomes via EAA (EXOEAA-PMO). EXOEAA-PMO elicited significantly greater muscle cell uptake, tissue accumulation and dystrophin expression than PMO in vitro and in vivo. Systemic administration of EXOEAA-PMO elicited therapeutic levels of dystrophin restoration and functional improvements in mdx mice. Altogether, our study demonstrates that EAA enables efficient loading of different nucleic acid drugs on exosomes, thus providing an easy and generalizable strategy for loading nucleic acid therapeutics on exosomes.

Development of Conformationally Restricted Negamycin Derivatives for Potent Readthrough Activity.

(+)-Negamycin, which is a dipeptide-like antibiotic containing a hydrazide structure, exhibits readthrough activity, resulting in the restoration of dystrophin in the mdx mouse model of Duchenne muscular dystrophy (DMD). In our previous structure-activity relationship study of negamycin, we found that its natural analogue 3-epi-deoxynegamycin (TCP-107), without antimicrobial activity, showed a higher readthrough activity than negamycin. In this study, we designed and synthesized cyclopropane-based conformationally restricted derivatives of TCP-107 and evaluated their readthrough activity in the cell-based reporter assay against a TGA-type mutation derived from DMD. As a result, a down-cis isomer, TCP-304, showed significant readthrough activity among the four isomers. Moreover, TCP-306, a derivative acylated by l-α-aminoundecanoic acid, possessed approximately 3 times higher activity than TCP-304. These down-cis derivatives showed dose-dependent readthrough activity and were effective for not only TGA but also TAG mutations. These results suggest that the conformational restriction of negamycin derivatives by the introduction of the cyclopropane ring is effective for an exhibition of potent readthrough activity.

Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.

Long-term dystrophin restoration supports development of splice correction therapy for DMD patients with exon 2 duplications

Long-term dystrophin restoration supports development of splice correction therapy for DMD patients with exon 2 duplications

P15 Full-length dystrophin restoration in multiple patient cell lines with DMD pseudoexons using AAV-delivered U7snRNA

P15 Full-length dystrophin restoration in multiple patient cell lines with DMD pseudoexons using AAV-delivered U7snRNA

Expression and function of Four AAV-based constructs for dystrophin restoration in the mdx mouse model of duchenne muscular dystrophy.

Robust expression of shortened, functional dystrophin provided impetus to develop adeno-associated virus (AAV)-based constructs for clinical application. Because several cassettes are being tested in clinical trials, this study compared efficacy of the following four shortened dystrophin-promoter combinations with implications for outcomes in clinical trials: MHCK7 or MCK promoter with a shortened dystrophin transgene containing the N-terminus and spectrin repeats R1, R2, R3 and R24 (rAAVrh74.MHCK7.micro-dystrophin and rAAVrh74.MCK.micro-dystrophin, respectively); shortened dystrophin construct containing the neuronal nitric oxide (nNOS) binding site (rAAVrh74.MHCK7.DV.mini-dystrophin); and shortened dystrophin containing the C-terminus (rAAVrh74.MHCK7.micro-dystrophin.Cterm). Functional and histological benefit were examined at 4 weeks following intramuscular delivery in mdx mice. rAAVrh74.MHCK7.micro-dystrophin provided the most robust transgene expression and significantly increased specific force output in the tibialis anterior muscle. Muscle environment was normalized (i.e., reductions in central nucleation), indicating functional and histological advantages of rAAVrh74.MHCK7.micro-dystrophin. Thus, promoter choice and transgene design are critical for optimal dystrophin expression/distribution for maximal functional improvement.

A highly sensitive and quantitative assay for dystrophin protein using Single Molecule Count Technology

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by progressive muscle loss caused by mutations in dystrophin, resulting in decreased dystrophin levels. Dystrophin protein expression is a biomarker used to evaluate treatments that restore patient dystrophin levels. Currently, a semiquantitative assay using western blotting, which normalizes dystrophin expression to that of a control population, is used for regulatory filing. However, the current methods are limited in terms of sensitivity, quantification, and reproducibility. To address this, a highly sensitive and quantitative sandwich immune assay using Single Molecule Counting technology was established, with recombinant dystrophin protein as the calibrator. Capture and detection antibodies were selected to detect full-length dystrophin. Using this optimized assay, dystrophin levels in muscle samples from Myotonic Dystrophy (n = 9) and DMD (n = 8) subjects were 93.2 ± 31.9 (range: 49.4–145.3) and 14.5 ± 6.8 (range: 6.18–22.6) fmol/total protein mg, respectively. The lowest concentration of dystrophin measured in the DMD samples was 5 times higher than that in the lower limit of quantitation, a level not detected by western blotting. These data indicate that this assay accurately and sensitively measured dystrophin protein and may be useful in clinical trials assessing dystrophin restoration therapies.

When Size Really Matters: The Eccentricities of Dystrophin Transcription and the Hazards of Quantifying mRNA from Very Long Genes.

At 2.3 megabases in length, the dystrophin gene is enormous: transcription of a single mRNA requires approximately 16 h. Principally expressed in skeletal muscle, the dystrophin protein product protects the muscle sarcolemma against contraction-induced injury, and dystrophin deficiency results in the fatal muscle-wasting disease, Duchenne muscular dystrophy. This gene is thus of key clinical interest, and therapeutic strategies aimed at eliciting dystrophin restoration require quantitative analysis of its expression. Approaches for quantifying dystrophin at the protein level are well-established, however study at the mRNA level warrants closer scrutiny: measured expression values differ in a sequence-dependent fashion, with significant consequences for data interpretation. In this manuscript, we discuss these nuances of expression and present evidence to support a transcriptional model whereby the long transcription time is coupled to a short mature mRNA half-life, with dystrophin transcripts being predominantly nascent as a consequence. We explore the effects of such a model on cellular transcriptional dynamics and then discuss key implications for the study of dystrophin gene expression, focusing on both conventional (qPCR) and next-gen (RNAseq) approaches.

Prevention of early-onset cardiomyopathy in Dmd exon 52–54 deletion mice by CRISPR-Cas9-mediated exon skipping

Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in Dmd that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD invivo.

AAV-vectored base editor trans-splicing delivers dystrophin repair

AAV-vectored base editor trans-splicing delivers dystrophin repair

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

Restoration of brain dystrophin using tricyclo-DNA ASOs restores neurobehavioral deficits in DMD mice

The Dilemma of Choice for Duchenne Patients Eligible for Exon 51 Skipping The European Experience.

Antisense oligonucleotide (ASO) mediated exon skipping aims to reframe dystrophin transcripts for patients with Duchenne muscular dystrophy (DMD). Currently 4 ASOs have been approved by the Food and Drug Administration targeting exon 45, 51 and 53 based on low level dystrophin restoration. Additional studies to confirm functional effects are ongoing. Furthermore, efforts are ongoing to increase muscle specific delivery of ASOs. Consequently, there are 5 clinical trials ongoing or planned for exon 51 skipping ASOs in Europe. While exon 51 skipping applies to the largest group of patients, DMD expert centers do not have sufficient numbers of patients or capacity to run all these trials in parallel. Even at a national level numbers may be too scarce. At the same time, some families now face the choice between participation in different clinical trials of exon 51 skipping, sometimes in addition to the choice of participating in a micro-dystrophin gene therapy trial. In this opinion paper, we outline the challenges, compare the different exon 51 skipping trials, and outline how different European centers and countries try to cope with running multiple trials in parallel for a small group of eligible patients.

The future of exon skipping for Duchenne muscular dystrophy.

Antisense oligonucleotide (ASO)-mediated exon skipping can restore the open reading frame of dystrophin transcripts for Duchenne muscular dystrophy (DMD) patients. This allows production of internally deleted dystrophin proteins as found in the later onset, less severely progressive Becker muscular dystrophy. Currently, four ASOs that induce exon skipping and dystrophin restoration are approved for the treatment of DMD by the Food and Drug Administration (FDAthe regulatory agencies of the USA and Japan). However, approval was based on restoration of very small amounts of dystrophin and the approved ASOs apply to only a subset of patients. This expert perspective evaluates ways to improve ASO efficiency that are currently in or close to clinical trials, as well as ways to improve applicability of this mutation-specific approach.

The unconditioned fear response in dystrophin-deficient mice is associated with adrenal and vascular function

Loss of function mutations in the gene encoding dystrophin elicits a hypersensitive fear response in mice and humans. In the dystrophin-deficient mdx mouse, this behaviour is partially protected by oestrogen, but the mechanistic basis for this protection is unknown. Here, we show that female mdx mice remain normotensive during restraint stress compared to a hypotensive and hypertensive response in male mdx and male/female wildtype mice, respectively. Partial dystrophin expression in female mdx mice (heterozygous) also elicited a hypertensive response. Ovariectomized (OVX) female mdx mice were used to explain the normotensive response to stress. OVX lowered skeletal muscle mass and lowered the adrenal mass and zona glomerulosa area (aldosterone synthesis) in female mdx mice. During a restraint stress, OVX dampened aldosterone synthesis and lowered the corticosterone:11-dehydrocorticosterone. All OVX-induced changes were restored with replacement of oestradiol, except that oestradiol lowered the zona fasciculata area of the adrenal gland, dampened corticosterone synthesis but increased cortisol synthesis. These data suggest that oestrogen partially attenuates the unconditioned fear response in mdx mice via adrenal and vascular function. It also suggests that partial dystrophin restoration in a dystrophin-deficient vertebrate is an effective approach to develop an appropriate hypertensive response to stress.

Partial restoration of brain dystrophin by tricyclo-DNA antisense oligonucleotides alleviates emotional deficits in mdx52 mice

The mdx52 mouse model recapitulates a frequent mutation profile associated with brain involvement in Duchenne muscular dystrophy. Deletion of exon 52 impedes expression of two dystrophins (Dp427, Dp140) expressed in brain, and is eligible for therapeutic exon-skipping strategies. We previously showed that mdx52 mice display enhanced anxiety and fearfulness, and impaired associative fear learning. In this study, we examined the reversibility of these phenotypes using exon 51 skipping to restore exclusively Dp427 expression in the brain of mdx52 mice. We first show that a single intracerebroventricular administration of tricyclo-DNA antisense oligonucleotides targeting exon 51 restores 5%-15% of dystrophin protein expression in the hippocampus, cerebellum, and cortex, at stable levels between 7 and 11week after injection. Anxiety and unconditioned fear were significantly reduced in treated mdx52 mice and acquisition of fear conditioning appeared fully rescued, while fear memory tested 24h later was only partially improved. Additional restoration of Dp427 in skeletal and cardiac muscles by systemic treatment did not further improve the unconditioned fear response, confirming the central origin of this phenotype. These findings indicate that some emotional and cognitive deficits associated with dystrophin deficiency may be reversible or at least improved by partial postnatal dystrophin rescue.

T Cell Responses to Dystrophin in a Natural History Study of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin, but many patients have rare revertant fibers that express dystrophin. The skeletal muscle pathology of DMD patients includes immune cell infiltration and inflammatory cascades. There are several strategies to restore dystrophin in skeletal muscles of patients, including exon skipping and gene therapy. There is some evidence that dystrophin restoration leads to a reduction in immune cells, but dystrophin epitopes expressed in revertant fibers or following genome editing, cell therapy, or microdystrophin delivery after adeno-associated viral gene therapy may elicit T cell production in patients. This may affect the efficacy of the therapeutic intervention, and potentially lead to serious adverse events. To confirm and extend previous studies, we performed annual enzyme- linked immunospot interferon-gamma assays on peripheral blood mononuclear cells from 77 pediatric boys with DMD recruited into a natural history study, 69 of whom (89.6%) were treated with corticosteroids. T cell responses to dystrophin were quantified using a total of 368 peptides spanning the entire dystrophin protein, organized into nine peptide pools. Peptide mapping pools were used to further localize the immune response in one positive patient. Six (7.8%) patients had a T cell-mediated immune response to dystrophin at at least one time point. All patients who had a positive result had been treated with corticosteroids, either prednisolone or prednisone. Our results show that ∼8% of DMD individuals in our cohort have a pre-existing T cell-mediated immune response to dystrophin, despite steroid treatment. Although these responses are relatively low level, this information should be considered a useful immunological baseline before undertaking clinical trials and future DMD studies. We further highlight the importance for a robust, reproducible standard operating procedure for collecting, storing, and shipping samples from multiple centers to minimize the number of inconclusive data.

Oligonucleotide Enhancing Compound Increases Tricyclo-DNA Mediated Exon-Skipping Efficacy in the Mdx Mouse Model.

Nucleic acid-based therapeutics hold great promise for the treatment of numerous diseases, including neuromuscular disorders, such as Duchenne muscular dystrophy (DMD). Some antisense oligonucleotide (ASO) drugs have already been approved by the US FDA for DMD, but the potential of this therapy is still limited by several challenges, including the poor distribution of ASOs to target tissues, but also the entrapment of ASO in the endosomal compartment. Endosomal escape is a well recognized limitation that prevents ASO from reaching their target pre-mRNA in the nucleus. Small molecules named oligonucleotide-enhancing compounds (OEC) have been shown to release ASO from endosomal entrapment, thus increasing ASO nuclear concentration and ultimately correcting more pre-mRNA targets. In this study, we evaluated the impact of a therapy combining ASO and OEC on dystrophin restoration in mdx mice. Analysis of exon-skipping levels at different time points after the co-treatment revealed improved efficacy, particularly at early time points, reaching up to 4.4-fold increase at 72 h post treatment in the heart compared to treatment with ASO alone. Significantly higher levels of dystrophin restoration were detected two weeks after the end of the combined therapy, reaching up to 2.7-fold increase in the heart compared to mice treated with ASO alone. Moreover, we demonstrated a normalization of cardiac function in mdx mice after a 12-week-long treatment with the combined ASO + OEC therapy. Altogether, these findings indicate that compounds facilitating endosomal escape can significantly improve the therapeutic potential of exon-skipping approaches offering promising perspectives for the treatment of DMD.

High-capacity adenovector delivery of forced CRISPR-Cas9 heterodimers fosters precise chromosomal deletions in human cells

Genome editing based on dual CRISPR-Cas9 complexes (multiplexes) permits removing specific genomic sequences in living cells leveraging research on functional genomics and genetic therapies. Delivering the required large and multicomponent reagents in a synchronous and stoichiometric manner remains, however, challenging. Moreover, uncoordinated activity of independently acting CRISPR-Cas9 multiplexes increases the complexity of genome editing outcomes. Here, we investigate the potential of fostering precise multiplexing genome editing using high-capacity adenovector particles (AdVPs) for the delivery of Cas9 ortholog fusion constructs alone (forced Cas9 heterodimers) or together with their cognate guide RNAs (forced CRISPR-Cas9 heterodimers). We demonstrate that the efficiency and accuracy of targeted chromosomal DNA deletions achieved by single AdVPs encoding forced CRISPR-Cas9 heterodimers is superior to that obtained when the various components are delivered separately. Finally, all-in-one AdVP delivery of forced CRISPR-Cas9 heterodimers triggers robust DMD exon 51 splice site excision resulting in reading frame restoration and selection-free detection of dystrophin in muscle cells derived from Duchenne muscular dystrophy patients. In conclusion, AdVPs promote precise multiplexing genome editing through the integrated delivery of forced CRISPR-Cas9 heterodimer components, which, in comparison with split conventional CRISPR-Cas9 multiplexes, engage target sequences in a more coordinated fashion.