Current Gene Therapy (v.12, #3)

The muscular dystrophies collectively represent a major health challenge, as few significant treatment options currently exist for any of these disorders. Recent years have witnessed a proliferation of novel approaches to therapy, spanning increased testing of existing and new pharmaceuticals, DNA delivery (both anti-sense oligonucleotides and plasmid DNA), gene therapies and stem cell technologies. While none of these has reached the point of being used in clinical practice, all show promise for being able to impact different types of muscular dystrophies. Our group has focused on developing direct gene replacement strategies to treat recessively inherited forms of muscular dystrophy, particularly Duchenne and Becker muscular dystrophy (DMD/BMD). Both forms of dystrophy are caused by mutations in the dystrophin gene and all cases can in theory be treated by gene replacement using synthetic forms of the dystrophin gene. The major challenges for success of this approach are the development of a suitable gene delivery shuttle, generating a suitable gene expression cassette able to be carried by such a shuttle, and achieving safe and effective delivery without elicitation of a destructive immune response. This review summarizes the current state of the art in terms of using adeno-associated viral vectors to deliver synthetic dystrophin genes for the purpose of developing gene therapy for DMD.

Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF and the consequent production of a shorter but functional dystrophin protein. This approach may lead to an effective disease modifying treatment for DMD and progress towards clinical application has been rapid. Less than a decade has passed between the first studies published in 1998 describing the use of AOs to modify the DMD gene in mice and the results of the first intramuscular proof of concept clinical trials. Whilst phase II and III trials are now underway, the heterogeneity of DMD mutations, efficient systemic delivery and targeting of AOs to cardiac muscle remain significant challenges. Here we review the current status of AO-mediated therapy for DMD, discussing the preclinical, clinical and regulatory hurdles and their possible solutions to expedite the translation of AO-mediated exon skipping therapy to clinic.

The hydrophobic plasma membrane constitutes an indispensable barrier for cells, allowing influx of essential molecules while preventing access to other macromolecules. Although pivotal for the maintenance of cells, the inability to cross the plasma membrane is one of the major obstacles toward current drug development. Oligonucleotides (ONs) are a group of substances that display great therapeutic potential to interfere with gene expression. Several classes of ONs have emerged either based on double stranded RNAs, such as short interfering RNAs that are utilized to confer gene silencing, or single stranded ONs of various chemistries for antisense targeting of small regulatory micro RNAs or mRNAs. In particular the use of splice switching oligonucleotides (SSOs) to manipulate alternative splicing, by targeting pre-mRNA, has proven to be a highly promising therapeutic strategy to treat various genetic disorders, including Duchenne muscular dystrophy and spinal muscular atrophy. Despite being efficient compounds to alter splicing patterns, their hydrophilic macromolecular nature prohibits efficient cellular internalization.Various chemical drug delivery vehicles have been developed aiming at improving the bioavailability of nucleic acid-based drugs. In the context of SSOs, one group of peptidebased delivery vectors, i.e. cell-penetrating peptides (CPPs), display extremely high potency. CPPs have a remarkable ability to convey various, otherwise impermeable, macromolecules across the plasma membrane of cells in a relatively non-toxic fashion. This review provides insight into the application of CPPs and ONs in gene regulation with particular focus on CPP-assisted delivery of therapeutic SSOs.

Splice-modulation therapy aiming at correcting genetic defects by molecular manipulation of the premessenger RNA is a promising novel therapeutic approach for genetic diseases. In recent years, these new RNA based strategies, mostly mediated by antisense oligonucleotides (AO), have demonstrated encouraging results for muscular dystrophies, a heterogeneous group of genetic disorders characterized by muscle weakness and wasting. In particular, the clinical evaluation of antisense-mediated exon-skipping for the treatment of Duchenne muscular dystrophy has shown convincing data and therefore raised hopes and expectations for neuromuscular disorders therapy. However, AO-mediated splicing modulation still faces major hurdles such as low efficacy in specific tissues, poor cellular uptake and relatively rapid clearance from circulation, which means repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, small nuclear RNAs (snRNA) have been used to shuttle the antisense sequences, offering the advantage of a correct subcellular localization with pre-mRNAs and the potential of a permanent correction when introduced into viral vectors. Here we review the recent progress in the development of snRNA mediated splicing modulation for muscular dystrophies, focusing on the advantages offered by this technology over classical AOs but also the challenges limiting their clinical application.

The Role of Stem Cells in Muscular Dystrophies by Mirella Meregalli (192-205).
Muscular dystrophies are heterogeneous neuromuscular disorders of inherited origin, including Duchenne muscular dystrophy (DMD). Cell-based therapies were used to promote muscle regeneration with the hope that the host cells repopulated the muscle and improved muscle function and pathology. Stem cells were preferable for therapeutic applications, due to their capacity of self-renewal and differentiative potential. In the last years, encouraging results were obtained with adult stem cells to treat muscular dystrophies. Adult stem cells were found into various tissues of the body and they were able to maintain, generate, and replace terminally differentiated cells within their own specific tissue because of cell turnover or tissue injury. Moreover, it became clear that these cells could participate into regeneration of more than just their resident organ. Here, we described multiple types of muscle and non muscle-derived myogenic stem cells, their characterization and their possible use to treat muscular dystrophies. We also underlined that most promising possibility for the management and therapy of DMD is a combination of different approaches, such as gene and stem cell therapy.

DMD is a devastatingly progressive muscle wasting disorder of childhood that significantly shortens life expectancy. Despite efforts to develop an effective therapy that dates back over a century, clinical interventions are still restricted to management of symptoms rather than a cure. The rationale to develop effective therapies changed in 1986 with the discovery of the dystrophin gene. Since then extensive research into both the molecular basis and pathophysiology of DMD has paved the way not only for development of strategies which aim to correct the primary defect, but also towards the identification of countless therapeutic targets with the potential to alleviate the downstream pathology. In addition to gene and cell-based therapies, which aim to deliver the missing gene and/or protein, an exciting spectrum of pharmacological approaches aimed at modulating therapeutic targets within DMD muscle cells through the use of small drugs are also being developed. This review presents promising pharmacological approaches aimed at targeting the primary defect, including suppression of nonsense mutations and functional compensation by upregulation of the dystrophin homologue, utrophin. Downstream of the primary membrane fragility, inflammation and fibrosis are reduced by blocking NF-κB, TGF-α and TGF-β, and free radical damage has been targeted using antioxidants and dietary/nutritional supplements. There are new hopes that ACE and PDE5 inhibitors can protect against skeletal as well as cardiac pathology, and modulating Ca2+ influx, NO, BMP, protein degradation and the mitochondrial permeability pore hold further promise in tackling the complex pathogenesis of this multifaceted disorder.

Since the discovery of the myostatin/ActRIIB signaling pathway 15 years ago, numerous strategies were developed to block its inhibitory function during skeletal muscle growth. Accumulating evidence demonstrates that abrogation of myostatin/ActRIIB signaling ameliorates pathology and function of dystrophic muscle in animal models for Duchenne muscular dystrophy (DMD). Therapeutic trials in healthy man and muscular dystrophy patients suggest feasibility of blockade strategies for potential clinical use. However, many key questions on the effect of myostatin/ActRIIB blockade remain unresolved; such as the underlying molecular mechanism that triggers muscle growth, the effect on muscle regeneration and adult muscle stem cell regulation and whether it causes long term metabolic alterations. Current therapeutic strategies aim to systemically abrogate myostatin/ActRIIB signaling. Although this ensures widespread effect on musculature, it also interferes with ActRIIB signaling in other tissues than skeletal muscle, thereby risking adverse effects. This review discusses current knowledge on myostatin/ActRIIB signaling and its potential value as a therapeutic target for DMD.