Objective: Phosphorodiamidate morpholino oligomers (PMOs) are synthetic nucleic acid analogs that can be designed to sequence-specifically block spliceosomes from binding to dystrophin pre-mRNA, resulting in omission of the targeted exon from the transcript and restoration of the reading frame with the goal of enabling synthesis of internally-shortened dystrophin. Background: DMD, a rare, X-linked genetic disease results in progressive muscle degeneration and premature death. DMD is primarily caused by whole exon deletions in the dystrophin gene resulting in a shift of the mRNA reading frame that prevents production of functional dystrophin protein. Design/Methods: As of June 3, 2016, 81 of 150 treated patients had received weekly eteplirsen for ≥1 year. Results: PMO eteplirsen received accelerated approval in the US for patients with a dystrophin gene mutation amenable to exon 51 skipping based on an increase in dystrophin in skeletal muscle in some patients. Mean dystrophin increases as measured by Western blot were observed following 180 weeks of treatment in the pivotal Phase II Studies 201/202 when compared to untreated DMD controls (N=11; +0.85%, p=0.007) and at Week 48 in Phase III Study PROMOVI when compared to baseline (N=12; +0.28%, p=0.008). Immunohistochemistry analysis at Week 180 in Study 201/202 also showed mean increases in dystrophin as measured by % dystrophin-positive fibers (N=11; +16.27%, p 4.5 years of treatment. Conclusions: Eteplirsen is the first exon skipping therapy approved for the treatment of Duchenne muscular dystrophy amenable to exon 51 skipping. Lessons learned from the eteplirsen clinical development program can aid in development of PMO therapies targeting additional exons. Study Supported by: Sarepta Therapeutics, Inc. Disclosure: Dr. Charleston has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. Schnell has received personal compensation for activities with Sarepta Therapeutics as a full time employee. Dr. Dworzak has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. Donoghue has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. Lynch has received personal compensation for activities with Sarepta Therapeutics, Inc. as an employee. Dr. Lewis has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Rodino-Klapac has nothing to disclose. Dr. Sahenk has nothing to disclose. Dr. Voss has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. DeAlwis has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. Frank has received personal compensation for activities with Sarepta Therapeutics, Inc. as an employee. Dr. Eliopoulos has received personal compensation for activities with Sarepta Therapeutics as an employee. Dr. Mendell has received personal compensation for activities with Sarepta Therapeutics, Inc.

Eteplirsen for the Treatment of Duchenne Muscular Dystrophy (DMD) (S42.001)

Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting ~1:5000 live male births. Following the identification of pathogenic variations in the dystrophin gene in 1986, the underlining genotype/phenotype correlations emerged and the role of the dystrophin protein was elucidated in skeletal, smooth, and cardiac muscles, as well as in the brain. When the dystrophin protein is absent or quantitatively or qualitatively modified, the muscle cannot sustain the stress of repeated contractions. Dystrophin acts as a bridging and anchoring protein between the sarcomere and the sarcolemma, and its absence or reduction leads to severe muscle damage that eventually cannot be repaired, with its ultimate substitution by connective tissue and fat. The advances of an understanding of the molecular pathways affected in DMD have led to the development of many therapeutic strategies that tackle different aspects of disease etiopathogenesis, which have recently led to the first successful approved orphan drugs for this condition. The therapeutic advances in this field have progressed exponentially, with second-generation drugs now entering in clinical trials as gene therapy, potentially providing a further effective approach to the condition.

Innovative Therapeutic Approaches for Duchenne Muscular Dystrophy.

In 1995, we were the first to propose antisense oligonucleotide (ASO)-mediated exon-skipping therapy for the treatment of Duchenne muscular dystrophy (DMD), a noncurable, progressive muscle-wasting disease. DMD is caused by deletion mutations in one or more exons of the DMD gene that shift the translational reading frame and create a premature stop codon, thus prohibiting dystrophin production. The therapy aims to correct out-of-frame mRNAs to produce in-frame transcripts by removing an exon during splicing, with the resumption of dystrophin production. As this treatment is recognized as the most promising, many extensive studies have been performed to develop ASOs that induce the skipping of DMD exons. In 2016, an ASO designed to skip exon 51 was first approved by the Food and Drug Administration, which accelerated studies on the use of ASOs to treat other monogenic diseases. The ease of mRNA editing by ASO-mediated exon skipping has resulted in the further application of exon-skipping therapy to nonmonogenic diseases, such as diabetes mellites. Recently, this precision medicine strategy was drastically transformed for the emergent treatment of only one patient with one ASO, which represents a future aspect of ASO-mediated exon-skipping therapy for extremely rare diseases. Herein, the invention of ASO-mediated exon-skipping therapy for DMD and the current applications of ASO-mediated exon-skipping therapies are reviewed, and future perspectives on this therapeutic strategy are discussed. This overview will encourage studies on ASO-mediated exon-skipping therapy and will especially contribute to the development of treatments for noncurable diseases.

Antisense Oligonucleotide-Mediated Exon-skipping Therapies: Precision Medicine Spreading from Duchenne Muscular Dystrophy

Significance Frameshift and nonsense mutations pose a major problem for disease therapeutic development. Eliminating these mutations from the messenger RNA by inducing exon skipping is a relatively unexplored treatment approach, though it has shown promise for some diseases. Here, we show that eliminating a common stop mutation associated with cystic fibrosis (CF), by inducing the skipping of the exon it is located in, results in a restoration of the open reading frame and recovers CFTR protein function in a manner expected to be therapeutic in CF patients who don’t currently have effective treatment options. These results are an important advancement for the CF community but also have implications for other diseases where terminating mutations are responsible for dysfunction. CFTR gene mutations that result in the introduction of premature termination codons (PTCs) are common in cystic fibrosis (CF). This mutation type causes a severe form of the disease, likely because of low CFTR messenger RNA (mRNA) expression as a result of nonsense-mediated mRNA decay, as well as the production of a nonfunctional, truncated CFTR protein. Current therapeutics for CF, which target residual protein function, are less effective in patients with these types of mutations due in part to low CFTR protein levels. Splice-switching antisense oligonucleotides (ASOs), designed to induce skipping of exons in order to restore the mRNA open reading frame, have shown therapeutic promise preclinically and clinically for a number of diseases. We hypothesized that ASO-mediated skipping of CFTR exon 23 would recover CFTR activity associated with terminating mutations in the exon, including CFTR p.W1282X, the fifth most common mutation in CF. Here, we show that CFTR lacking the amino acids encoding exon 23 is partially functional and responsive to corrector and modulator drugs currently in clinical use. ASO-induced exon 23 skipping rescued CFTR expression and chloride current in primary human bronchial epithelial cells isolated from a homozygote CFTR-W1282X patient. These results support the use of ASOs in treating CF patients with CFTR class I mutations in exon 23 that result in unstable CFTR mRNA and truncations of the CFTR protein.

https://www.pnas.org/content/pnas/119/3/e2114886119.full.pdf

Open reading frame correction using splice-switching antisense oligonucleotides for the treatment of cystic fibrosis

Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abn...

Optogenetic modeling of human neuromuscular circuits in Duchenne muscular dystrophy with CRISPR and pharmacological corrections.

Exon skipping compounds restoring the dystrophin transcript reading frame have received regulatory approval for Duchenne muscular dystrophy (DMD). Recently, focus shifted to developing compounds to...

https://www.tandfonline.com/doi/pdf/10.1080/14712598.2021.1832462

Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy

Genome-wide association studies (GWAS) have been highly informative in discovering disease-associated loci but are not designed to capture all structural variations in the human genome. Using long-read sequencing data, we discovered widespread structural variation within SINE-VNTR-Alu (SVA) elements, a class of great ape-specific transposable elements with gene-regulatory roles, which represents a major source of structural variability in the human population. We highlight the presence of structurally variable SVAs (SV-SVAs) in neurological disease–associated loci, and we further associate SV-SVAs to disease-associated SNPs and differential gene expression using luciferase assays and expression quantitative trait loci data. Finally, we genetically deleted SV-SVAs in the BIN1 and CD2AP Alzheimer's disease–associated risk loci and in the BCKDK Parkinson's disease–associated risk locus and assessed multiple aspects of their gene-regulatory influence in a human neuronal context. Together, this study reveals a novel layer of genetic variation in transposable elements that may contribute to identification of the structural variants that are the actual drivers of disease associations of GWAS loci.

/pdf/a-hidden-layer-of-structural-variation-in-transposable-27zcbcgj.pdf

Anne-Fleur E Schneider

Papers

Developments in reading frame restoring therapy approaches for Duchenne muscular dystrophy

A hidden layer of structural variation in transposable elements reveals potential genetic modifiers in human disease-risk loci