Search Results for: duchenne muscular dystrophy ips stem cell treatment

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January 13, 2020

We expect that a satellite cell with the corrected DMD gene would quite quickly and continuously propagate the edited gene throughout the muscle tissue, said Prof. Amy Wagers, who leads the research. (Photo credit: Jon Chase/Harvard Staff Photographer.)

Cambridge, Mass. January 13, 2020 Harvard University stem cell researchers led by Amy Wagers, PhD, are embarking on a major study of Duchenne muscular dystrophy (DMD). Supported by research funding from Sarepta Therapeutics, under a multi-year collaboration agreement coordinated by Harvards Office of Technology Development (OTD), the project aims to use in-vivo genome editing, in mouse models of DMD, to fully and precisely restore the function of the dystrophin protein, which is crucial for proper muscular growth and development. Approaches validated by this work may point the way to an eventual therapeutic strategy to reverse DMD in humans.

Duchenne muscular dystrophy is a genetic disease caused by the lack of a protein called dystrophin that normally helps to support the structural integrity of muscle fibers, including those in the heart. Without the dystrophin protein, cells are weaker and degenerate more quickly. Over time, affected individuals boys, typically, as it is a recessive X-linked disorder lose their capacity to move independently.

Its really a devastating disease; it robs young boys of their capacity to be young boys, said Wagers, who is the Forst Family Professor of Stem Cell and Regenerative Biology, Co-Chair of the Department of Stem Cell & Regenerative Biology, and an Executive Committee Member of Harvard Stem Cell Institute. Though it is early days, Im hopeful that through this work we may identify and validate new avenues for therapy to completely rescue the proper expression and function of the dystrophin protein and regenerate healthy muscle tissue.

Researchers worldwide have pursued a variety of promising approaches such as cell and gene therapies, small-molecule therapies, and others to lessen or prevent the disease and improve patients quality of life.

The strategy pursued by the Wagers Lab at Harvard aims to fully correct the genetic template for dystrophin at its source, in the DNA of stem cells (satellite cells) that create and regenerate muscle cells. Combining cutting-edge CRISPR/Cas9 genome editing technologies with a deep knowledge of stem cell science and regenerative biology, this approach if successful might offer a permanent restoration of muscular function.

In skeletal muscle, muscle fibers are terminally post-mitotic, meaning they cannot divide and they cannot reproduce themselves, Wagers explains. If you lose muscle fibers, the only way to produce new muscle is from stem cells, specifically the satellite cells. The satellite cells are self-renewing, self-repairing, and ready to spring into action to create new muscle fibers. So we expect that a satellite cell with the corrected DMD gene would quite quickly and continuously propagate the edited gene throughout the muscle tissue.

At present, research conducted in mice has shown promising results. In June, the Wagers Lab published the results of editing stem cells in vivo, demonstrating that stem cell genes can be edited in living systems, not only in a dish. In that work, Wagers and her team delivered genome editing molecules to the cells using adeno-associated viruses (AAVs). Her lab has also successfully used gene editing in heart, muscle, and satellite cells to partially restore the function of the DMD gene that encodes dystrophin, by chopping out faulty sequences of code that are disrupting the proper reading frame.

The new stem-cell approach pursued in collaboration with Sarepta would build on these achievements and use more precise genome editing approaches, in animal models of DMD, to entirely replace genetic mutations in the DMD gene with correctly encoded sequences. The project will also explore alternate delivery methods and strategies to mitigate immune effects of in vivo genome editing.

This ambitious project will benefit greatly from the resources and insights of a company with deep clinical experience in the development of therapeutics for muscular dystrophy, said Vivian Berlin, Managing Director of Strategic Partnerships at Harvard OTD. Preclinical discoveries by Harvard researchers may open entirely new possibilities for lifesaving treatments in the long run, offering much-needed hope to patients and families in the future. Were grateful to be able to sustain the important momentum already established in Prof. Wagers lab, through this collaboration.

As we work to bring forward new treatments for patients with DMD, Sarepta is excited to support Prof. Wagers and her lab to accelerate the development of a gene editing approach, which has shown significant potential in early studies, said Louise Rodino-Klapac, Sareptas Senior Vice President of Gene Therapy. This multi-year collaboration is part of Sareptas broader commitment to pursuing all therapeutic modalities and advancing our scientific understanding of gene editing in order to maximize the potential of this approach to help patients.

Under the terms of the agreement between Harvard and Sarepta, the company will have the exclusive option to license any arising intellectual property for the purpose of developing products to prevent and treat human disease. As with any research agreement facilitated by OTD, the right of academic and other not-for-profit researchers to use the technology in further scholarly work is preserved.

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Muscular dystrophy collaboration aims to correct muscle stem cells' DNA - Harvard Office of Technology Development

1. Introduction

Neuromuscular disease is a very broad term that encompasses many diseases and aliments that either directly, via intrinsic muscle pathology, or indirectly, via nerve pathology, impair the functioning of the muscles. Neuromuscular diseases affect the muscles and/or their nervous control and lead to problems with movement. Many are genetic; sometimes, an immune system disorder can cause them. As they have no cure, the aim of clinical treatment is to improve symptoms, increase mobility and lengthen life. Some of them affect the anterior horn cell, and are classified as acquired (e.g. poliomyelitis) and hereditary (e.g. spinal muscular atrophy) diseases. SMA is a genetic disease that attacks nerve cells, called motor neurons, in the spinal cord. As a consequence of the lost of the neurons, muscles weakness becomes to be evident, affecting walking, crawling, breathing, swallowing and head and neck control. Neuropathies affect the peripheral nerve and are divided into demyelinating (e.g. leucodystrophies) and axonal (e.g. porphyria) diseases. Charcot-Marie-Tooth (CMT) is the most frequent hereditary form among the neuropathies and its characterized by a wide range of symptoms so that CMT-1a is classified as demyelinating and CMT-2 as axonal (Marchesi & Pareyson, 2010). Defects in neuromuscular junctions cause infantile and non-infantile Botulism and Myasthenia Gravis (MG). MG is a antibody-mediated autoimmune disorder of the neuromuscular junction (NMJ) (Drachman, 1994; Meriggioli & Sanders, 2009). In most cases, it is caused by pathogenic autoantibodies directed towards the skeletal muscle acetylcholine receptor (AChR) (Patrick & Lindstrom, 1973) while in others, non-AChR components of the postsynaptic muscle endplate, such as the muscle-specific receptor tyrosine kinase (MUSK), might serve as targets for the autoimmune attack (Hoch et al., 2001). Although the precise origin of the autoimmune response in MG is not known, genetic predisposition and abnormalities of the thymus gland such as hyperplasia and neoplasia could have an important role in the onset of the disease (Berrih et al., 1984; Roxanis et al., 2001).

Several diseases affect muscles: they are classified as acquired (e.g. dermatomyositis and polymyositis) and hereditary (e.g. myotonic disorders and myopaties) forms. Among the myopaties, muscular dystrophies are characterized by the primary wasting of skeletal muscle, caused by mutations in the proteins that form the link between the cytoskeleton and the basal lamina (Cossu & Sampaolesi, 2007). Mutations in the dystrophin gene cause severe form of hereditary muscular diseases; the most common are Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD). DMD patients suffer for complete lack of dystrophin that causes progressive degeneration, muscle wasting and death into the second/third decade of life. Beside, BMD patients show a very mild phenotype, often asymptomatic primarily due to the expression of shorter dystrophin mRNA transcripts that maintain the coding reading frame. DMD patients muscles show absence of dystrophin and presence of endomysial fibrosis, small fibers rounded and muscle fiber degeneration/regeneration. Untreated, boys with DMD become progressively weak during their childhood and stop ambulation at a mean age of 9 years, later with corticosteroid treatment (12/13 yrs). Proximal weakness affects symmetrically the lower (such as quadriceps and gluteus) before the upper extremities, with progression to the point of wheelchair dependence. Eventually distal lower and then upper limb weakness occurs. Weakness of neck flexors is often present at the beginning, and most patients with DMD have never been able to jump. Wrist and hand muscles are involved later, allowing the patients to keep their autonomy in transfers using a joystick to guide their wheelchair. Musculoskeletal contractures (ankle, knees and hips) and learning difficulties can complicate the clinical expression of the disease. Besides this weakness distribution in the same patient, a deep variability among patients does exist. They could express a mild phenotype, between Becker and Duchenne dystrophy, or a really severe form, with the loss of deambulation at 7-8 years. Confinement to a wheelchair is followed by the development of scoliosis, respiratory failure and cardiomyopathy. In 90% of people death is directly related to chronic respiratory insufficiency (Rideau et al., 1983). The identification and characterization of dystrophin gene led to the development of potential treatments for this disorder (Bertoni, 2008). Even if only corticosteroids were proven to be effective on DMD patient (Hyser and Mendell, 1988), different therapeutic approaches were attempted, as described in detail below (see section 7).

The identification and characterization of the genes whose mutations caused the most common neuromuscular diseases led to the development of potential treatments for those disorders. Gene therapy for neuromuscular disorders embraced several concepts, including replacing and repairing a defective gene or modifying or enhancing cellular performance, using gene that is not directly related to the underlying defect (Shavlakadze et al., 2004). As an example, the finding that DMD pathology was caused by mutations in the dystrophin gene allowed the rising of different therapeutic approaches including growth-modulating agents that increase muscle regeneration and delay muscle fibrosis (Tinsley et al., 1998), powerful antisense oligonucleotides with exon-skipping capacity (Mc Clorey et al., 2006), anti-inflammatory or second-messenger signal-modulating agents that affect immune responses (Biggar et al., 2006), agents designed to suppress stop codon mutations (Hamed, 2006). Viral and non-viral vectors were used to deliver the full-length - or restricted versions - of the dystrophin gene into stem cells; alternatively, specific antisense oligonucleotides were designed to mask the putative splicing sites of exons in the mutated region of the primary RNA transcript whose removal would re-establish a correct reading frame. In parallel, the biology of stem cells and their role in regeneration were the subject of intensive and extensive research in many laboratories around the world because of the promise of stem cells as therapeutic agents to regenerate tissues damaged by disease or injury (Fuchs and Segre, 2000; Weissman, 2000). This research constituted a significant part of the rapidly developing field of regenerative biology and medicine, and the combination of gene and cell therapy arose as one of the most suitable possibility to treat degenerative disorders. Several works were published in which stem cell were genetically modified by ex vivo introduction of corrective genes and then transplanted in donor dystrophic animal models.

Stem cells received much attention because of their potential use in cell-based therapies for human disease such as leukaemia (Owonikoko et al., 2007), Parkinsons disease (Singh et al., 2007), and neuromuscular disorders (Endo, 2007; Nowak and Davies, 2004). The main advantage of stem cells rather than the other cells of the body is that they can replenish their numbers for long periods through cell division and, they can produce a progeny that can differentiate into multiple cell lineages with specific functions (Bertoni, 2008). The candidate stem cell had to be easy to extract, maintaining the capacity of myogenic conversion when transplanted into the host muscle and also the survival and the subsequent migration from the site of injection to the compromise muscles of the body (Price et al., 2007). With the advent of more sensitive markers, stem cell populations suitable for clinical experiments were found to derive from multiple region of the body at various stage of development. Numerous studies showed that the regenerative capacity of stem cells resided in the environmental microniche and its regulation. This way, it could be important to better elucidate the molecular composition cytokines, growth factors, cell adhesion molecules and extracellular matrix molecules - and interactions of the different microniches that regulate stem cell development (Stocum, 2001).

Several groups published different works concerning adult stem cells such as muscle-derived stem cells (Qu-Petersen et al., 2002), mesoangioblasts (Cossu and Bianco, 2003), blood- (Gavina et al., 2006) and muscle (Benchaouir et al., 2007)-derived CD133+ stem cells. Although some of them are able to migrate through the vasculature (Benchaouir et al., 2007; Galvez et al., 2006; Gavina et al., 2006) and efforts were done to increase their migratory ability (Lafreniere et al., 2006; Torrente et al., 2003a), poor results were obtained.

Embryonic and adult stem cells differ significantly in regard to their differentiation potential and in vitro expansion capability. While adult stem cells constitute a reservoir for tissue regeneration throughout the adult life, they are tissue-specific and possess limited capacity to be expanded ex vivo. Embryonic Stem (ES) cells are derived from the inner cell mass of blastocyst embryos and, by definition, are capable of unlimited in vitro self-renewal and have the ability to differentiate into any cell type of the body (Darabi et al., 2008b). ES cells, together with recently identified iPS cells, are now broadly and extensively studied for their applications in clinical studies.

Embryonic stem cells are pluripotent cells derived from the early embryo that are characterized by the ability to proliferate over prolonged periods of culture remaining undifferentiated and maintaining a stable karyotype (Amit and Itskovitz-Eldor, 2002; Carpenter et al., 2003; Hoffman and Carpenter, 2005). They are capable of differentiating into cells present in all 3 embryonic germ layers, namely ectoderm, mesoderm, and endoderm, and are characterized by self-renewal, immortality, and pluripotency (Strulovici et al., 2007).

hESCs are derived by microsurgical removal of cells from the inner cell mass of a blastocyst stage embryo (Fig. 1). The ES cells can be also obtained from single blastomeres. This technique creates ES cells from a single blastomere directly removed from the embryo bypassing the ethical issue of embryo destruction (Klimanskaya et al., 2006). Although maintaining the viability of the embryo, it has to be determined whether embryonic stem cell lines derived from a single blastomere that does not compromise the embryo can be considered for clinical studies. Cell Nuclear Transfer (SCNT): Nuclear transfer, also referred to as nuclear cloning, denotes the introduction of a nucleus from an adult donor cell into an enucleated oocyte to generate a cloned embryo (Wilmut et al., 2002).

ESCs differentiation. Differentiation potentiality of human embryonic stem cell lines. Human embryonic stem cell pluripotency is evaluated by the ability of the cells to differentiate into different cell types.

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