Duchenne muscular dystrophy (DMD) has been a major target for gene therapy development for nearly 30 years. DMD is among the most common genetic diseases, and isolation of the defective gene (DMD, or ...dystrophin) was a landmark discovery, as it was the first time a human disease gene had been cloned without knowledge of the protein product. Despite tremendous obstacles, including the enormous size of the gene and the large volume of muscle tissue in the human body, efforts to devise a treatment based on gene replacement have advanced steadily through the combined efforts of dozens of labs and patient advocacy groups. Progress in the development of DMD gene therapy has been well documented in Molecular Therapy over the past 20 years and will be reviewed here to highlight prospects for success in the imminent human clinical trials planned by several groups.
Duchenne muscular dystrophy (DMD) is one of the most common human genetic disorders. Cloning of the DMD gene preceded the human genome project and established DMD as an early gene therapy target. We summarize progress using AAV vectors for bodywide dystrophin gene delivery, an approach rapidly moving into clinical trials.
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 is a genome-editing technology1,2 that utilizes archaeal and bacterial Cas9 nucleases to introduce double-stranded breaks in ...DNA at targeted sites. These breaks can be used to remove, replace, or add pieces of DNA. While not the first genome editor, CRISPR-Cas9 is efficient and cost-effective because cutting is guided by a strand of RNA rather than a protein. The potential uses in health care are plentiful, from disrupting dominant genes that cause cancer3 to repairing mutated genes that cause genetic diseases, such as muscular dystrophy4. Therapeutic approaches based on this technology fill the preclinical pipeline, and rely on the use of viral vectors to deliver the Cas9 gene and guide RNA to a gene of interest. However, concerns regarding the safety and efficacy of CRISPR-Cas9 use in gene therapy remain. A pre-print released prior to peer review has recently underlined the question of whether immunological responses to Cas9 may negatively impact its clinical use5. Here we discuss the implications of this finding for the application of CRISPR/Cas in gene therapy.
Gene replacement therapies utilizing adeno-associated viral (AAV) vectors hold great promise for treating Duchenne muscular dystrophy (DMD). A related approach uses AAV vectors to edit specific ...regions of the DMD gene using CRISPR/Cas9. Here we develop multiple approaches for editing the mutation in dystrophic mdx
mice using single and dual AAV vector delivery of a muscle-specific Cas9 cassette together with single-guide RNA cassettes and, in one approach, a dystrophin homology region to fully correct the mutation. Muscle-restricted Cas9 expression enables direct editing of the mutation, multi-exon deletion or complete gene correction via homologous recombination in myogenic cells. Treated muscles express dystrophin in up to 70% of the myogenic area and increased force generation following intramuscular delivery. Furthermore, systemic administration of the vectors results in widespread expression of dystrophin in both skeletal and cardiac muscles. Our results demonstrate that AAV-mediated muscle-specific gene editing has significant potential for therapy of neuromuscular disorders.
A Boost for Muscle with Gene Therapy Chamberlain, Jeffrey S
The New England journal of medicine,
2022-Mar-24, Letnik:
386, Številka:
12
Journal Article
Abstract
Muscular dystrophy (MD) is a group of progressive genetic diseases affecting the musculature that are characterized by inflammatory infiltrates, necrosis and connective tissue and fat ...replacement of the affected muscles. Unfortunately, treatments do not exist for the vast majority of MD patients. Adeno-associated viral vector (AAV)-based gene therapy is thus emerging as a potential treatment for many types of MD. Treatments strategies based on AAV are being adapted for replacement of mutant disease-causing genes, knockdown of dominant disease-causing genes using antisense oligonucleotides or inhibitory RNAs, delivery of gene editing tools such as clustered regularly interspaced short palindromic repeats/Cas9 and effecting alterations in pre-mRNA splicing and by manipulating expression levels of modifier genes. Translational and clinical trial work focused on these types of AAV treatments for Duchenne MD, various limb girdle MDs, myotonic dystrophy 1, facioscapulohumeral MD, dysferlinopathies and congenital MDs are discussed here, with a focus on recent studies, pre-clinical large animal work and many promising ongoing and upcoming AAV clinical trials.
Adeno-associated virus (AAV) vectors have shown promising results in preclinical models, but the genomic consequences of transduction with AAV vectors encoding CRISPR-Cas nucleases is still being ...examined. In this study, we observe high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea. Genome-wide AAV mapping in mouse brain shows no overall increase of AAV integration except at the CRISPR/Cas9 target site. To allow detailed characterization of integration events we engineer a miniature AAV encoding a 465 bp lambda bacteriophage DNA (AAV-λ465), enabling sequencing of the entire integrated vector genome. The integration profile of AAV-465λ in cultured cells display both full-length and fragmented AAV genomes at Cas9 on-target sites. Our data indicate that AAV integration should be recognized as a common outcome for applications that utilize AAV for genome editing.
ABSTRACT
Duchenne muscular dystrophy (DMD) is a devastating muscle disorder that affects 1 in 3,500 boys. Despite years of research and considerable progress in understanding the molecular mechanism ...of the disease and advancement of therapeutic approaches, there is no cure for DMD. The current treatment options are limited to physiotherapy and corticosteroids, and although they provide a substantial improvement in affected children, they only slow the course of the disorder. On a more optimistic note, more recent approaches either significantly alleviate or eliminate muscular dystrophy in murine and canine models of DMD and importantly, many of them are being tested in early phase human clinical trials. This review summarizes advancements that have been made in viral and nonviral gene therapy as well as stem cell therapy for DMD with a focus on the replacement and repair of the affected dystrophin gene. Muscle Nerve 47: 649–663, 2013
Recombinant adeno-associated virus (rAAV) holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the muscular dystrophies. Given the ...variety of applications and tissue types toward which these vectors may be targeted, an understanding of rAAV transduction is crucial for the effective application of therapy. rAAV transduction mechanisms have been the subject of much study, resulting in a body of knowledge relating to events from virus-cell attachment through to vector genome conformation in the target cell nucleus. Instead of utilizing one mechanism in each phase of vector transduction, rAAV appears to employ multiple possible pathways toward transgene expression, in part dependent on rAAV serotype, dose, and target cell type. Once inside the nucleus, the rAAV genome exists in a predominantly episomal form; therefore, nondividing cells tend to be most stably transduced. However, rAAV has a low frequency of integration into the host cell genome, often in or near genes, and can be associated with host genome mutations. This review describes the current understanding of the mechanisms and rate-limiting steps involved in rAAV transduction.
Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. ...However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (μUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-μUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for μUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed μUtrn. As such, we found that μUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike μUtrn, μdystrophin (μDys) expression did not show this fiber type preference. Interestingly, μUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & μUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.
Gene editing is often touted as a permanent method for correcting mutations, but its long-term benefits in Duchenne muscular dystrophy (DMD) may depend on sufficiently high editing efficiencies to ...halt muscle degeneration. Here, we explored the persistence of dystrophin expression following recombinant adeno-associated virus serotype 6 (rAAV6):CRISPR-Cas9-mediated multi-exon deletion/reframing in systemically injected 2- and 11-week-old dystrophic mice and show that induction of low dystrophin levels persists for several months in cardiomyocytes but not in skeletal muscles, where myofibers remain susceptible to necrosis and regeneration. Whereas gene-correction efficiency in both muscle types was enhanced with increased ratios of guide RNA (gRNA)-to-nuclease vectors, obtaining high dystrophin levels in skeletal muscles via multi-exon deletion remained challenging. In contrast, when AAV-microdystrophin was codelivered with editing components, long-term gene-edited dystrophins persisted in both muscle types. These results suggest that the high rate of necrosis and regeneration in skeletal muscles, compared with the relative stability of dystrophic cardiomyocytes, caused the rapid loss of edited genomes. Consequently, stable dystrophin expression in DMD skeletal muscles will require either highly efficient gene editing or the use of cotreatments that decrease skeletal muscle degeneration.
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Bengtsson et al. show that the efficacy of dystrophin gene-editing in skeletal, but not cardiac, muscle depends on the rate of muscle necrosis/regeneration. The authors further show that muscle stabilization, here via codelivery of microdystrophin, significantly improves long-term outcomes of therapeutic gene editing in a mouse model of Duchenne muscular dystrophy.
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