Linda Popplewell

Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study

Summary Background Mutations that disrupt the open reading frame and prevent full translation of DMD, the gene that encodes dystrophin, underlie the fatal X-linked disease Duchenne muscular dystrophy. Oligonucleotides targeted to splicing elements (splice switching oligonucleotides) in DMD pre-mRNA can lead to exon skipping, restoration of the open reading frame, and the production of functional dystrophin in vitro and in vivo, which could benefit patients with this disorder. Methods We did a single-blind, placebo-controlled, dose-escalation study in patients with DMD recruited nationally, to assess the safety and biochemical efficacy of an intramuscular morpholino splice-switching oligonucleotide (AVI-4658) that skips exon 51 in dystrophin mRNA. Seven patients with Duchenne muscular dystrophy with deletions in the open reading frame of DMD that are responsive to exon 51 skipping were selected on the basis of the preservation of their extensor digitorum brevis (EDB) muscle seen on MRI and the response of cultured fibroblasts from a skin biopsy to AVI-4658. AVI-4658 was injected into the EDB muscle; the contralateral muscle received saline. Muscles were biopsied between 3 and 4 weeks after injection. The primary endpoint was the safety of AVI-4658 and the secondary endpoint was its biochemical efficacy. This trial is registered, number NCT00159250. Findings Two patients received 0·09 mg AVI-4658 in 900 μL (0·9%) saline and five patients received 0·9 mg AVI-4658 in 900 μL saline. No adverse events related to AVI-4658 administration were reported. Intramuscular injection of the higher-dose of AVI-4658 resulted in increased dystrophin expression in all treated EDB muscles, although the results of the immunostaining of EDB-treated muscle for dystrophin were not uniform. In the areas of the immunostained sections that were adjacent to the needle track through which AVI-4658 was given, 44–79% of myofibres had increased expression of dystrophin. In randomly chosen sections of treated EDB muscles, the mean intensity of dystrophin staining ranged from 22% to 32% of the mean intensity of dystrophin in healthy control muscles (mean 26·4%), and the mean intensity was 17% (range 11–21%) greater than the intensity in the contralateral saline-treated muscle (one-sample paired t test p=0·002). In the dystrophin-positive fibres, the intensity of dystrophin staining was up to 42% of that in healthy muscle. We showed expression of dystrophin at the expected molecular weight in the AVI-4658-treated muscle by immunoblot. Interpretation Intramuscular AVI-4658 was safe and induced the expression of dystrophin locally within treated muscles. This proof-of-concept study has led to an ongoing systemic clinical trial of AVI-4658 in patients with DMD. Funding UK Department of Health.

β-catenin as a potential key target for tumor suppression.

β‐catenin is a multifunctional protein identified to be pivotal in embryonic patterning, organogenesis and adult homeostasis. It plays a critical structural role in mediating cadherin junctions and is also an essential transcriptional co‐activator in the canonical Wnt pathway. Evidence has been documented that both the canonical Wnt pathway and cadherin junctions are deregulated or impaired in a plethora of human malignancies. In the light of this, there has been a recent surge in elucidating the mechanisms underlying the etiology of cancer development from the perspective of β‐catenin. Here, we focus on the emerging roles of β‐catenin in the process of tumorigenesis by discussing novel functions of old players and new proteins, mechanisms identified to mediate or interact with β‐catenin and the most recently unraveled clinical implications of β‐catenin regulatory pathways toward tumor suppression.

Design of Phosphorodiamidate Morpholino Oligomers (PMOs) for the Induction of Exon Skipping of the Human DMD Gene

Duchenne muscular dystrophy (DMD) is caused by out-of-frame mutations of the human DMD gene. Antisense oligonucleotides (AOs) have previously been used to skip additional exons that border the deletions such that the reading frame is restored and internally truncated, but functional, dystrophin expressed. We have designed phosphorodiamidate morpholino oligomer (PMO) AOs to various exons of the human dystrophin gene. PMOs were designed to have their target sites overlapping areas of open RNA structure, as defined by hybridization-array analysis, and likely exonic splicing enhancer (ESE)/silencer sites on the target RNA. The ability of each PMO to produce exon skipping was tested in vitro in normal human skeletal muscle cells. Retrospective analysis of design parameters used and PMO variables revealed that active PMOs were longer, bound to their targets more strongly, had their target sites closer to the acceptor splice site of the exon, overlapped areas of open conformation (as defined by the hybridization or the RNA secondary structure prediction software), and could interfere with the binding of certain SR proteins. No other parameter appeared to show significant association to PMO-skipping efficacy. No design tool is strong enough in isolation; however, if used in conjunction with other significant parameters it can aid AO design.

Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases

The ability to specifically engineer the genome of living cells at precise locations using rare-cutting designer endonucleases has broad implications for biotechnology and medicine, particularly for functional genomics, transgenics and gene therapy. However, the potential impact of chromosomal context and epigenetics on designer endonuclease-mediated genome editing is poorly understood. To address this question, we conducted a comprehensive analysis on the efficacy of 37 endonucleases derived from the quintessential I-CreI meganuclease that were specifically designed to cleave 39 different genomic targets. The analysis revealed that the efficiency of targeted mutagenesis at a given chromosomal locus is predictive of that of homologous gene targeting. Consequently, a strong genome-wide correlation was apparent between the efficiency of targeted mutagenesis (≤0.1% to ∼6%) with that of homologous gene targeting (≤0.1% to ∼15%). In contrast, the efficiency of targeted mutagenesis or homologous gene targeting at a given chromosomal locus does not correlate with the activity of individual endonucleases on transiently transfected substrates. Finally, we demonstrate that chromatin accessibility modulates the efficacy of rare-cutting endonucleases, accounting for strong position effects. Thus, chromosomal context and epigenetic mechanisms may play a major role in the efficiency rare-cutting endonuclease-induced genome engineering.

Gene therapy for muscular dystrophy: current progress and future prospects

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there is no effective treatment but new gene-based therapies are currently being developed with particular noted advances in using conventional gene replacement strategies, RNA-based approaches, or cell-based gene therapy with a main focus on Duchenne muscular dystrophy (DMD). DMD is the most common and severe form of muscular dystrophy and current treatments are far from adequate. However, genetic and cell-based therapies, in particular exon skipping induced by antisense strategies, and corrective gene therapy via functionally engineered dystrophin genes hold great promise, with several clinical trials ongoing. Proof-of-concept of exon skipping has been obtained in animal models, and most recently in clinical trials; this approach represents a promising therapy for a subset of patients. In addition, gene-delivery-based strategies exist both for antisense-induced reading frame restoration, and for highly efficient delivery of functional dystrophin mini- and micro-genes to muscle fibres in vivo and muscle stem cells ex-vivo. In particular, AAV-based vectors show efficient systemic gene delivery to skeletal muscle directly in vivo, and lentivirus-based vectors show promise of combining ex vivo gene modification strategies with cell-mediated therapies.

Gene Correction of a Duchenne Muscular Dystrophy Mutation by Meganuclease-Enhanced Exon Knock-In

Duchenne muscular dystrophy (DMD) is a severe inherited, muscle-wasting disorder caused by mutations in the DMD gene. Gene therapy development for DMD has concentrated on vector-based DMD minigene transfer, cell-based gene therapy using genetically modified adult muscle stem cells or healthy wild-type donor cells, and antisense oligonucleotide-induced exon-skipping therapy to restore the reading frame of the mutated DMD gene. This study is an investigation into DMD gene targeting-mediated correction of deletions in human patient myoblasts using a target-specific meganuclease (MN) and a homologous recombination repair matrix. The MN was designed to cleave within DMD intron 44, upstream of a deletion hotspot, and integration-competent lentiviral vectors expressing the nuclease (LVcMN) were generated. MN western blotting and deep gene sequencing for LVcMN-induced non-homologous end-joining InDels (microdeletions or microinsertions) confirmed efficient MN expression and activity in transduced DMD myoblasts. A homologous repair matrix carrying exons 45-52 (RM45-52) was designed and packaged into integration-deficient lentiviral vectors (IDLVs; LVdRM45-52). After cotransduction of DMD myoblasts harboring a deletion of exons 45 to 52 with LVcMN and LVdRM45-52 vectors, targeted knock-in of the RM45-52 region in the correct location in DMD intron 44, and expression of full-length, correctly spliced wild-type dystrophin mRNA containing exons 45-52 were observed. This work demonstrates that genome surgery on human DMD gene mutations can be achieved by MN-induced locus-specific genome cleavage and homologous recombination knock-in of deleted exons. The feasibility of human DMD gene repair in patient myoblasts has exciting therapeutic potential.

New developments in the use of gene therapy to treat Duchenne muscular dystrophy

Introduction: Duchenne muscular dystrophy (DMD) is a lethal X-linked inherited disorder characterised by progressive muscle weakness, wasting and degeneration. Although the gene affected in DMD was identified over 25 years ago, there is still no effective treatment. Areas covered: Here we review some of the genetic-based strategies aimed at amelioration of the DMD phenotype. A number of Phase II/III clinical trials of antisense oligonucleotide-induced exon skipping for restoration of the open reading frame (ORF) of the DMD gene have recently been completed. The potential strategies for overcoming the hurdles that appear to prevent exon skipping becoming an effective treatment for DMD currently are discussed. Expert opinion: The applicability of exon skipping as a therapy to DMD is restricted and the development of alternative strategies that are more encompassing is needed. The rapid pre-clinical advances that are being made in the field of adeno-associated virus (AAV)-based delivery of micro-dystrophin would address this. The obstacles to be faced with gene replacement strategies would include the need for high viral titres, efficient muscle targeting and avoidance of immune response to vector and transgene. The new emerging field of gene editing could potentially provide permanent correction of the DMD gene and the feasibility of such an approach to DMD is discussed.

Genetic Therapeutic Approaches for Duchenne Muscular Dystrophy

Despite an expansive wealth of research following the discovery of the DMD gene 25 years ago, there is still no curative treatment for Duchenne muscular dystrophy. However, there are currently many promising lines of research, including cell-based therapies and pharmacological reagents to upregulate dystrophin via readthrough of nonsense mutations or by upregulation of the dystrophin homolog utrophin. Here we review genetic-based therapeutic strategies aimed at the amelioration of the DMD phenotype. These include the reintroduction of a copy of the DMD gene into an affected tissue by means of a viral vector; correction of the mutated DMD transcript by antisense oligonucleotide-induced exon skipping to restore the open reading frame; and direct modification of the DMD gene at a chromosomal level through genome editing. All these approaches are discussed in terms of the more recent advances, and the hurdles to be overcome if a comprehensive and effective treatment for DMD is to be found. These hurdles include the need to target all musculature of the body. Therefore any potential treatment would need to be administered systemically. In addition, any treatment needs to have a long-term effect, with the possibility of readministration, while avoiding any potentially detrimental immune response to the vector or transgene.

Delivery of AAV2/9-Microdystrophin Genes Incorporating Helix 1 of the Coiled-Coil Motif in the C-Terminal Domain of Dystrophin Improves Muscle Pathology and Restores the Level of α1-Syntrophin and α-Dystrobrevin in Skeletal Muscles of mdx Mice

Duchenne muscular dystrophy is a severe X-linked inherited muscle wasting disorder caused by mutations in the dystrophin gene. Adeno-associated virus (AAV) vectors have been extensively used to deliver genes efficiently for dystrophin expression in skeletal muscles. To overcome limited packaging capacity of AAV vectors (<5 kb), truncated recombinant microdystrophin genes with deletions of most of rod and carboxyl-terminal (CT) domains of dystrophin have been developed. We have previously shown the efficiency of mRNA sequence-optimized microdystrophin (ΔR4-23/ΔCT, called MD1) with deletion of spectrin-like repeat domain 4 to 23 and CT domain in ameliorating the pathology of dystrophic mdx mice. However, the CT domain of dystrophin is thought to recruit part of the dystrophin-associated protein complex, which acts as a mediator of signaling between extracellular matrix and cytoskeleton in muscle fibers. In this study, we extended the ΔR4-23/ΔCT microdystrophin by incorporating helix 1 of the coiled-coil motif in the CT domain of dystrophin (MD2), which contains the α1-syntrophin and α-dystrobrevin binding sites. Intramuscular injection of AAV2/9 expressing CT domain-extended microdystrophin showed efficient dystrophin expression in tibialis anterior muscles of mdx mice. The presence of the CT domain of dystrophin in MD2 increased the recruitment of α1-syntrophin and α-dystrobrevin at the sarcolemma and significantly improved the muscle resistance to lengthening contraction-induced muscle damage in the mdx mice compared with MD1. These results suggest that the incorporation of helix 1 of the coiled-coil motif in the CT domain of dystrophin to the microdystrophins will substantially improve their efficiency in restoring muscle function in patients with Duchenne muscular dystrophy.

Comparative analysis of antisense oligonucleotide sequences targeting exon 53 of the human DMD gene: Implications for future clinical trials.

Duchenne muscular dystrophy (DMD) is caused by the lack of functional dystrophin protein, most commonly as a result of a range of out-of-frame mutations in the DMD gene. Modulation of pre-mRNA splicing with antisense oligonucleotides (AOs) to restore the reading frame has been demonstrated in vitro and in vivo, such that truncated but functional dystrophin is expressed. AO-induced skipping of exon 51 of the DMD gene, which could treat 13% of DMD patients, has now progressed to clinical trials. We describe here the methodical, cooperative comparison, in vitro (in DMD cells) and in vivo (in a transgenic mouse expressing human dystrophin), of 24 AOs of the phosphorodiamidate morpholino oligomer (PMO) chemistry designed to target exon 53 of the DMD gene, skipping of which could be potentially applicable to 8% of patients. A number of the PMOs tested should be considered worthy of development for clinical trial.