"Inherited Muscle Disease: Gene Therapy". In: Encyclopedia of Life

Inherited Muscle Disease: Gene Therapy Terence Partridge, Imperial College School of Medicine, London, UK

Advanced article Article Contents . Introduction . Clinical Features . Gene Therapy in Model Systems

Muscular dystrophies have been an area of special interest for genetic therapies. The abundance of skeletal muscle tissue in the body presents particular problems for this form of treatment.

. Gene Transfer Systems Applicable to Muscle Disease . Clinical Applications . Problems and Future Directions

doi: 10.1002/9780470015902.a0005753.pub2

Introduction Genetic or quasi-genetic therapies are potentially applicable to both acquired and inherited diseases of skeletal muscle but this article reflects a general preoccupation in research on the latter. However, this distinction of objectives has become blurred in recent times by the demonstration that some pathological consequences of inherited muscular dystrophies appear to be ameliorated by strategies designed to prevent muscle wasting, and it is likely, in the future, that broader socio-economic interests in combatting the age-associated loss of muscle size and strength will come to dominate the incentives for research in this topic. Although there is a growing interest in the application of genetic or quasi-genetic therapies to acquired diseases of skeletal muscle, a general preoccupation with inherited defects of this tissue is reflected in this article. However, the distinction of objectives has been blurred in recent times by the demonstration that the pathological consequences of inherited muscular dystrophies may, in some instances, derive benefit from general strategies designed to combat muscle wasting, and the widespread quality of life concerns associated with age-related muscle wasting may, in the future, become a major driving force in this research area. As a class, the muscular dystrophies have been an area of special interest for genetic therapies, conceivably because the gene responsible for Duchenne muscular dystrophy (DMD) was the first disease gene to be identified by positional cloning (Koenig et al., 1987). Partly as a consequence of this, there has been a disproportionate flow of discovery of further monogenic muscular dystrophies. A concerted movement to discover genetic therapies for dystrophies has been driven by the fact that these diseases blight the lives of children and young adults and have proved refractory to conventional therapies. DMD and its milder allelic variant, Becker muscular dystrophy (BMD), are X-linked recessive conditions that epitomize the main problems of gene therapy; for the dystrophin gene responsible is by far the largest and among the most complex in the human genome, making it difficult to fit into some prospective viral vectors. Moreover, the principal target tissue, skeletal muscle, is the most abundant cell type in the body and is widely distributed, making it a difficult target for comprehensive gene therapy. At the same time, the dystrophin gene offers some therapeutic

prospects, discussed below, that are not available with other genes. DMD will therefore be used as the central example, reference being made to important features of other genetic muscle diseases as appropriate.

Clinical Features The muscular dystrophies are the most severe and common genetic diseases of skeletal muscle. They are characterized by muscle wasting, associated with degeneration and regeneration of skeletal muscle and usually by progressive accumulation of collagenous scar tissue and fat. Cardiac muscle is often affected too. Genetic defects of muscle that do not feature conspicuous degeneration and regeneration are termed ‘myopathies’ but this distinction is not absolute. DMD is the most common fatal X-linked disease, estimated to affect approximately 1 in 3500 male live births. It is largely asymptomatic in female carriers but follows a relentless course in affected boys. Classically, clumsiness and weakness are noted in early childhood with loss of the ability to walk by 12 years of age and death from cardiac or respiratory disease occurring in the late teens or early 20s. This progression can be delayed by palliative treatment of skeletal deformities, by respiratory support, and in recent years by administration of glucocorticoids. Since one-third of cases occur as new mutations, there is no prospect of eliminating the disease by genetic counselling, and there will always be a substantial number of boys in need of therapy. In normal muscle, the 427 kDa protein dystrophin underlies the plasmalemmal membrane of all skeletal and cardiac muscle fibres as a key member of a complex of proteins that spans the plasmalemma, linking subsurface cytoplasmic actin to laminin in the basement membrane that is closely applied to the outside of each fibre (Figure 1). In the absence of dystrophin, the other members of the complex fail to assemble correctly and are lost or present in diminished amounts at the plasmalemmal surface. In boys with DMD, dystrophin is completely, or almost completely, absent; but in the milder allelic variant, BMD, aberrant forms of dystrophin are found, commonly in less than normal amounts. Most DMD mutations are deletions or duplications predicted to disrupt the reading frame of

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Inherited Muscle Disease: Gene Therapy

Figure 1 Hypothetical view of the dystrophin-associated complex of proteins and glycoproteins at the surface of the muscle fibre. Dystrophin, the protein that is lacking in Duchenne muscular dystrophy and is defective in the milder Becker muscular dystrophy, appears to be the key protein required for effective assembly of this complex, elucidation of which has proved an interesting example of the interaction between positional cloning and biochemical investigation. Mutations in the genes for the various sarcoglycans have been found to underlie some of the limb-girdle muscular dystrophies (LGMD): a-Sarcoglycan – LGMD 2D, bSarcoglycan – LGMD 2E, g-Sarcoglycan – LGMD 2C, d-Sarcoglycan – LGMD 2F. A large proportion of congenital muscular dystrophy cases have proved to involve mutations in the a-chain of merosin, the link of this complex into the basement membrane. The muscle-specific isoform of neural nitric oxide synthase, mNOS, and the dystrophin-like protein, dystroglycan, are also linked into this complex and disruption of this complex may compromise their functions and perhaps contribute to some aspects of the pathology of the dystrophinopathies. Other dystrophies are ascribed to proteins that do not appear to be part of this complex, some associated with the cell surface, some with the nuclear membranes and some with the sarcoplasm (for review, see Cohn and Campbell (2000) Molecular basis of muscular dystrophies. Muscle and Nerve 23: 1456–1471).

the messenger ribonucleic acid (mRNA) transcript, but some splice site mutations and nonsense mutations have also been described. In BMD, the mutation is usually such as to specify an mRNA transcript in which the codon triplets retain an intact open reading frame and can be translated into a 2

protein carrying a small defect such as a deletion or duplication (Monaco et al., 1988). Importantly, some very mild BMD patients have large deletions of the middle ‘rod repeat region’ of dystrophin, indicating that the N- and C-terminal regions carry most of the important function (England et al., 1990). This has been exploited in the design



of ‘minidystrophins’ that will fit into viral vectors and also in devising methods of producing partially functional dystrophins from the mutant endogenous gene by skipping sections that disrupt translation. Other muscular dystrophies are regularly identified, many with defects in other components of the dystrophinassociated complex, but some have no obvious link to this system. Other monogenic defects of muscle that do not conform to a dystrophic clinical phenotype are also being identified at the rate of several per year. These are catalogued and updated monthly on the Neuromuscular Disorders home page (see Web Links).

Gene Therapy in Model Systems Almost all of the data we have on the various vectors and strategies described below were generated on the dystrophic mdx mouse. Some are now being repeated on the dystrophic golden retriever muscular dystrophy (GRMD) dog but cost precludes the use of this animal for primary testing. In recent years, new mouse models have been generated by targeting the genes that have been identified as being associated with human myopathies. These are being increasingly used as models of these diseases. The mdx mouse has a severe primary pathology, in that from the onset of the disease at 2–3 weeks almost all of its muscle fibres degenerate and regenerate by a few months of age. It is thus useful for assessing any effects of putative gene therapies directed at opposing the pathological events that are the immediate consequence of the lack of dystrophin. Features downstream of the primary pathology, especially the ultimate clinical sequellae, are more likely to differ between species and in both the mouse and the dog, should be viewed circumspectly as precise models of the human disease.

Gene Transfer Systems Applicable to Muscle Disease Strategies The simplest basic strategy for inherited muscle disease is to complement the defective gene by introducing a sound copy into the muscle cells. This was the objective of most initial proposals for genetic therapies for inherited muscular diseases. In principle, this is a valid strategy for recessive diseases but dominant genetic diseases, such as myotonic dystrophy, would require additional measures to nullify the ill effects of the defective gene. The most theoretically satisfactory approach would be to repair the defective gene by gene-targeting techniques. The most effective, though seemingly capricious, method for achieving this is the use of chimaeric deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) constructs, which have been demonstrated to repair point mutations at low

overall frequency in the dystrophin gene of the mdx mouse (Bertoni et al., 2003). Falling slightly short of this ideal are two strategies that have arisen in the past few years for rescuing a partially functional protein product from the mRNA transcript of the mutant dystrophin gene. One, currently in clinical trial (see Web Links), is applicable only to nonsense mutations that generate a stop codon within exons and involves use of substances that raise the frequency with which translation is continued through the stop codon to produce a protein that closely resembles dystrophin except for the single abnormal amino acid that is inserted at the mutation site (Welch et al., 2007). The second, also in preliminary trial in patients (see Web Links) relies on the fact that the majority of DMD mutations lie in parts of the gene that do not encode the most functionally important 3’ and 5’ domains of the dystrophin protein. It involves the use of oligonucleotide sequences complementary to those at important sites involved in splicing, to mask the splice sites of particular exons and thus prevent their inclusion in the eventual mRNA transcript. By excluding exons that carry nonsense mutations or whose elimination would restore the reading frame of the transcript, it is possible, in theory, to convert a Duchenne to a milder Becker dystrophy (Aartsma-Rus et al., 2006). This approach has been validated in the mdx mouse (Dunckley et al., 1998; Lu et al., 2003) and is particularly promising because some types of oligonucleotide analogue can be delivered to the muscles in effective doses via the blood and are fully resistant to metabolic degradation (Alter et al., 2006). It is in a preliminary experimental stage in human patients (see Web Links). A more genetically based approach to this same idea is to generate, intracellularly, a sequence that masks exon splice sites by introducing an expression plasmid into the muscle fibres by means of a recombinant adeno-associated vector (see below). This has been accomplished by use of a modified U7 snRNA that is involved normally in splicing of histone mRNAs and so is well maintained within the cell. By replacing the normal U7 recognition sequence with one designed to mask sites involved in splicing of the murine dystrophin exon 23 it has been proved possible to normalize the functional deficits in the mdx mouse (Goyenvalle et al., 2004). Gene ‘redundancy’, where products of separate genes overlap one another partially in function, has led to proposals for ameliorating the consequences of naturally occurring genetic defects. For example, the lack of dystrophin in mdx mice has been shown to be functionally compensated by the transgene for its autosomal homologue utrophin (Tinsley et al., 1998). Similar over-expression of a7b1integrin in muscle has been found to reduce the severity of muscle disease in the mdx–Utr–/– mouse (Burkin et al., 2001). To convert this idea to a useful therapy requires the discovery of agents that drive over-expression of the replacement gene in skeletal muscle without deleteriously affecting expression of other genes. Such agents might be simple organic chemicals or more complex substances


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designed to imitate transcription factors involved in the control of the gene in question (Khurana and Davies, 2003). A specific advantage of this strategy is that the body should be tolerant of the substitute protein and should not mount an immune response against it.

have been shown to be capable of significantly ameliorating the dystrophies arising from a-sarcoglycan deficiency in the mouse, corresponding to human limb-girdle muscular dystrophy 2D (Galvez et al., 2006) and dystrophin deficiency in the dog (Sampaolesi et al., 2006).

Vectors

Direct transfection of muscle with dystrophin expression plasmids

Nature of the target Apart from the large size of the dystrophin gene, a particular problem for gene therapy of muscle is that it is a large and diffuse target that has few receptors that would facilitate efficient and specific gene delivery. A variety of methods have been used to deliver dystrophin to muscle, each of which has its own particular advantages and disadvantages. Skeletal and cardiac muscle are both ‘end cells’ in which the nuclei of the fully differentiated cells do not divide, although skeletal muscle has ‘satellite cells’ which do proliferate and fuse together to repair, replace and provide material for growth of the muscle fibres. Two features of this system are advantageous for gene therapy. First, the longevity of individual muscle fibres favours persistent expression of introduced constructs even where these remain episomal and do not integrate into the genome of the muscle fibre nuclei. Second, the muscle precursor cells themselves provide a mechanism for introducing genetic material into muscle fibres, forming the basis of ‘myoblast transfer’ and latterly for ‘stem cell’ therapies.

Transplantation of myogenic cells The first proposals for gene therapy on muscle were based on the use of myogenic satellite cells as gene vectors (Partridge et al., 1989). In principle, myogenic cells derived from a normal donor could be grafted or the patient’s own myogenic cells could be genetically corrected and reimplanted. The former plan involves greater immunological problems but is technically simpler. To date, trials of myoblast transplantation in DMD volunteers have given only modest local restoration of dystrophin (Skuk et al., 2006). Cell-transplantation strategies were revived recently by the demonstration that circulating stem cells are able to enter damaged skeletal and cardiac muscle and to engage in myogenesis (Ferrari et al., 1998; Gussoni et al., 2006) with the implied promise of a systemic delivery of myogenic cells to the widespread sites of muscle damage in DMD. However, up to now this has proven to be a rare event, demonstrable only with very sensitive markers and too inefficient to be of practical value. Yet, another variant of this idea has arisen over the past few years from the isolation and propagation of cells derived from the small blood vessels. These cells, called mesoanioblasts, have a limited proliferative capacity and it remains in doubt whether they are stem cells, in the sense of forming part of the normal mechanism of repair and maintenance of skeletal muscle. But they do possess the important quality of being deliverable to muscle with moderate efficiency via intra-arterial perfusion and 4

Skeletal and cardiac muscle are surprisingly susceptible to transfection by direct injection of naked DNA expression plasmids. These appear to enter sporadic fibres in the region of injection and to be expressed very persistently as episomes. It has been found possible, by use of high pressure perfusion, to transduce appreciable proportions of muscle fibres of an isolated limb with naked DNA expression plasmids (Hagstrom et al., 2004). This approach, avoids any problems of immune response against proteins associated with the vectors.

Retroviral vectors Recombinant replication-defective retroviruses were among the first vectors to be tested for achieving genetic alteration of skeletal muscle with the idea of transducing proliferating satellite cells that would then fuse into muscle fibres in the process of repairing them. These vectors appear to be locally effective in introducing genes into damaged skeletal muscle and the transduced satellite cells also form an enduring genetically corrected reservoir for future repair (Fassati and Bresolin, 2000). Despite these advantages the overall efficiency of this approach has been too low to promote its further development.

Lentiviral vectors Use of recombinant lentivirus vectors to introduce a nonmuscle gene into skeletal muscle, were not, initially, very encouraging but advances in understanding and in production technology have provided progressive improvements and the capacity of this class of vector to integrate into the genomes of both dividing and nondividing cells makes them attractive for use in muscle where not only the satellite cells (MacKenzie et al., 2005) but also potentially the myonuclei of muscle fibres and cardiomyocytes can be transduced.

Adenoviral vectors During the early phase of gene therapy research, recombinant adenoviruses were heavily favoured as potential vectors of the dystrophin gene. Their main attractions were their wide range of cell targets, the high titres attainable and the potential for carrying a full-length dystrophin gene. In postnatal or regenerating mouse muscle they are highly effective in converting fibres near the injection site. However, it has proved difficult to infect muscle efficiently in older animals. There are also problems of immune responses both against proteins encoded via the viral genome and against the coat proteins (Yuasa et al., 2002). Progressive refinements have eliminated the former problem



(Chen et al., 1997) but the coat protein remains an issue if multiple reapplication of the vector is envisaged.

Herpes vectors Recombinant herpes viral vectors have much the same advantages and disadvantages as adenoviral vectors. They have a potential capacity for large genes but are not efficient at transducing older muscle (Huard et al., 1997) and have been reported to be cytotoxic.

Adenoassociated viral (AAV) vectors Initially, difficulties of production at high titre and of purification from the helper adenovirus, delayed the application of AAV-based vectors. In addition, the size of construct they can carry is very restricted, requiring the design of ‘microdystrophins’ lacking almost the entire rod domain. On the other hand, they appear to be natural residents of skeletal muscle not associated with any known pathology and they transduce mature skeletal muscle fibres very efficiently and persistently, with little or no immune response against the transgenic protein (Wang et al., 2000). There is however a strong antibody response against viral proteins that presents a problem for repeated delivery. Some variants have been shown to be very effective in delivering a micro-dystrophin gene to body-wide muscles of the dystrophic mouse, with associated beneficial effects (Gregorevic et al., 2006). A trial of local delivery into DMD volunteers by intramuscular injection of an optimized microdystrophin expression plasmid with a chimaeric AAV (Rabinowitz et al., 2002) vector is currently underway (see Web Links).

Clinical Applications At present, the only clinical tests of gene therapy reported have been on myoblast transplantation. These have produced either no detectable effect or trivial increases in the amount of dystrophin present at the graft site (Gussoni et al., 1992). One centre claiming significant improvements in physiological function in patients grafted with normal myoblasts (Law et al., 1997), was heavily criticized by the food and drug administration (FDA) and no longer appears to be recruiting for treatment of DMD. A study has been proposed on the use of AAV vectors to introduce the missing sarcoglycan genes into the muscles of patients suffering from limb-girdle muscular dystrophies (Trial of AAV therapy for gamma-sarcoglycanopathy http://www.genethon.fr/index.php?id=49&L=1). Human trials of quasi-genetic therapies are currently being conducted on the use of PTC124 to enhance read-through of nonsense mutations in the dystrophin gene (web-site) and 2 preliminary trials on use of antisense oligonucleotides to restore reading frame in the dystrophin gene (web-sites).

Problems and Future Directions The overwhelming problems for gene therapy in skeletal muscle are those of obtaining dispersed efficient targeting of the vector to muscles throughout the body. Of the delivery systems for replacement genes, efficient bodywide deliver seems plausibly supported by evidence only in the case of AAV-microdystrophins and the use of allogeneic mesoangioblasts as cellular vectors. For cell-based delivery, there is some interest in enhancing the efficiency of targeting and myogenic conversion of circulating stem cells in sites of muscle regeneration (Dezawa et al., 2005). Procedures that promote fluid efflux from the microvascular bed into the interstitial space have been reported to give dispersed delivery of adenovirus vectors and naked plasmids in small rodents and in nonhuman primates (Zhang et al., 2001) but, to date, no human trials employing such methods have been reported. The problems of immune response to new proteins or parts of proteins are common to most attempts at gene replacement therapies although adenoassociated viral vectors are reported to largely avoid this problem in skeletal muscle perhaps because they do not transduce antigenpresenting cells. Strategies that rely on the activation or rescue of endogenous genes by means of small diffusible reagents have the advantage with respect to both an easier systemic delivery and the lower likelihood of an immune response against the elicited protein. Although most attempts at gene therapy are directed at the primary genetic defect, many genetic diseases, including muscular dystrophies, exert much of their clinical harm via downstream pathological mechanisms, such as inflammation, fibrosis and exhaustion of progenitor cells that normally replenish the tissue. Each of these mechanisms may also be regarded as a target of gene therapy, for instance by expression of growth factors, cytokines or inhibitors of these agents, in such a way as to counteract harmful processes and promote beneficial ones. This same rationale could, of course, also be applied in alleviating the pathogenesis of acquired myopathies. See also: Duchenne Muscular Dystrophy (DMD) Gene; Muscular Dystrophies

References Aartsma-Rus A, Kaman WE, Weij R et al. (2006) Exploring the frontiers of therapeutic exon skipping for Duchenne muscular dystrophy by double targeting within one or multiple exons. Molecular Therapy. 14(3): 401–407. Alter J, Lou F, Rabinowitz A et al. (2006) Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nature Medicine 12(2): 175–177. Bertoni C, Lau C, Rando AT et al. (2003) Restoration of dystrophin expression in mdx muscle cells by chimeraplast-mediated exon skipping. Human Molecular Genetics 12(10): 1087–1099.


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Burkin DJ, Wallace GQ, Nicol KJ, Kaufman DJ and Kaufman SJ (2001) Enhanced expression of the a7b1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. Journal of Cell Biology 152: 1207–1218. Chen HH, Mack LM, Kelly R et al. (1997) Persistence in muscle of an adenoviral vector that lacks all viral genes. Proceedings of the National Academy of Sciences of the USA 94: 1645–1650. Dezawa M, Ishikawa H, Itokazu Y et al. (2005) Bone marrow stromal cells generate muscle cells and repair muscle degeneration. Science 309(5732): 314–317. Dunckley MG, Manoharan M, Villiet P, Eperon IC and Dickson G (1998) Modification of splicing in the dystrophin gene in cultured Mdx muscle cells by antisense oligoribonucleotides. Human Molecular Genetics 7: 1083–1090. England SB, Nicholson LVB, Johnson MA et al. (1990) Very mild muscular dystrophy associated with deletion of 46% of dystrophin. Nature 343: 180–182. Fassati A and Bresolin N (2000) Retroviral vectors for gene therapy of Duchenne muscular dystrophy. Neurological Sciences 21(suppl. 5): S925–S927. Ferrari G, Cusella-De Angelis G, Coletta M et al. (1998) Muscle regeneration by bone marrow-derived myogenic precursors. Science 279: 1528–1530. Galvez BG, Sampaolesi M, Brunelli S et al. (2006) Complete repair of dystrophic skeletal muscle by mesoangioblasts with enhanced migration ability. Journal of Cell Biology 174(2): 231–243. Goyenvalle A, Vulin A, Fougerousse F et al. (2004) Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping. Science 306(5702): 1796–1799. Gregorevic P, Allen JM, Harper SQ et al. (2006) rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nature Medicine 12(7): 787–789. Gussoni E, Pavlath GK, Lanctot AM et al. (1992) Normal dystrophin transcripts detected in Duchenne muscular dystrophy patients after myoblast transplantation. Nature 356: 435–438. Gussoni E, Soneoka Y, Strickland CD et al. (1999) Dystrophin expression in the mdx mouse restored by stem cell transplantation. Nature 410: 390–394. Hagstrom JE, Hegge J, Zhang G et al. (2004) A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs. Molecular Therapy 10(2): 386–398. Huard J, Krisky D, Oligino T et al. (1997) Gene transfer to muscle using herpes simplex virus-based vectors. Neuromuscular Disorders 7: 299–313. Khurana TS and Davies KE (2003) Pharmacological strategies for muscular dystrophy. Nature Reviews Drug Discovery 2(5): 379–390. Koenig M, Hoffman EP, Bertelson CJ et al. (1987) Complete cloning of the Duchenne muscular dystrophy (DMD) cDNA and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 50: 509–517. Law PK, Goodwin TG, Fang Q et al. (1997) Human gene therapy with myoblast transfer. Transplantation Proceedings 29: 2234–2237. Lu QL, Mann CJ, Lou F et al. (2003) Functional amounts of dystrophin produced by skipping the mutated exon in the mdx dystrophic mouse. Nature Medicine 9(8): 1009–1014. MacKenzie TC, Kobinger GP, Kootstra NA et al. (2005) Transduction of satellite cells after prenatal intramuscular

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administration of lentiviral vectors. Journal of Genetic Medicine 7(1): 50–58. Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H and Kunkel LM (1988) An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 2: 90–95. Partridge TA, Morgan JE, Coulton GR et al. (1989) Conversion of mdx myofibres from dystrophin-negative to -positive by injection of normal myoblasts. Nature 337(6203): 176–179. Rabinowitz JE, Rolling F, Li C et al. (2002) Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. Journal of Virology 76(2): 791–801. Sampaolesi M, Blot S, D’Antona G et al. (2006) Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs. Nature 444(7119): 574–579. Skuk D, Goulet M, Roy B et al. (2006) Dystrophin expression in muscles of duchenne muscular dystrophy patients after highdensity injections of normal myogenic cells. Journal of Neuropathology & Experimental Neurology 65(4): 371–386. Stedman H, Wilson JM, Finke R, Kleckner AL and Mendell J (2000) Phase I clinical trial utilizing gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma- or delta-sarcoglycan gene delivered with intramuscular instillations of adenoassociated vectors. Human Gene Therapy 11: 777–790. Tinsley J, Deconinck N, Fisher R et al. (1998) Expression of fulllength utrophin prevents muscular dystrophy in mdx mice. Nature Medicine 4: 1441–1444. Wang B, Li J and Xiao X (2000) Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proceedings of the National Academy of Sciences of the USA 97: 13714–13719. Welch EM, Barton ER, Zhuo J et al. (2007) PTC124 is a novel therapeutic for the treatment of genetic disorders caused by nonsense mutations. Nature 447(7140): 87–91. Yuasa K, Sakamoto M, Miyagoe-Suzuki Y et al. (2002) Adenoassociated virus vector-mediated gene transfer into dystrophindeficient skeletal muscles evokes enhanced immune response against the transgene product. Gene Therapy 9(23): 1576–1588. Zhang G, Budker V, Williams P, Subbotin V and Wolff JA (2001) Efficient expression of naked DNA delivered intraarterially to limb muscles of nonhuman primates. Human Gene Therapy 12: 427–438.

Further Reading Ahn AH and Kunkel LM (1993) The structural and functional diversity of dystrophin. Nature Genetics 3: 283–291. Bartlett RJ, Stockinger S, Denis MM et al. (2000) In vivo targeted repair of a point mutation in the canine dystrophin gene by a chimeric RNA/DNA oligonucleotide. Nature Biotechnology 18: 615–622. Barton-Davis ER, Cordier L, Shoturma DI, Leland SE and Sweeney HL (1999) Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice. Journal of Clinical Investigation 104: 375–381. Barton-Davis ER, Shoturma DI, Musaro A, Rosenthal N and Sweeney HL (1998) Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle



function. Proceedings of the National Academy of Sciences of the USA 95(26): 15603–15607. Bittner RE, Scho¨fer C, Weipoltshammer K et al. (1999) Recruitment of bone-marrow-derived cells by skeletal and cardiac muscle in adult dystrophic mdx mice. Anatomy and Embryology 199: 391–396. Budker V, Zhang G, Danko I, Williams P and Wolff J (1998) The efficient expression of intravascularly delivered DNA in rat muscle. Gene Therapy 5: 272–276 Links . Cohn RD and Campbell KP (2000) Molecular basis of muscular dystrophies. Muscle and Nerve 23: 1456–1471. Dunckley MG, Wells DJ, Walsh FS and Dickson G (1993) Direct retroviral-mediated transfer of a dystrophin minigene into mdx mouse muscle in vivo. Human Molecular Genetics 2: 717–723. Dystrophin (muscular dystrophy, Duchenne and Becker types) (DMD); Locus ID: 1756. LocusLink: http://www.ncbi.nlm. nih.gov/LocusLink/LocRpt.cgi?l=1756 Dystrophin (muscular dystrophy, Duchenne and Becker types) (DMD); MIM number: 300377. OMIM: http://www.ncbi.nlm. nih.gov/htbin-post/Omim/dispmim?300377 Exon skipping with 2’O-methyl phosphorothioate antisense oligonucleotides to restore open reading frame in the mutated dystrophin gene http://hdl.handle.net/1887/604 Feero WG, Rosenblatt JD, Huard J et al. (1997) Viral gene delivery to skeletal muscle: insights on maturation- dependent loss of fiber infectivity for adenovirus and herpes simplex type 1 viral vectors. Human Gene Therapy 8: 371–380. Greelish JP, Su LT, Lankford EB et al. (1999) Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector. Nature Medicine 5: 439–443.

Hartigan-O’Connor D, Amalfitano A and Chamberlain JS (1999) Improved production of gutted adenovirus in cells expressing adenovirus preterminal protein and DNA polymerase. Journal of Virology 73: 7835–7841. Jiang ZL, Reay D, Kreppel F et al. (2001) Local high-capacity adenovirus-mediated mCTLA4Ig and mCD40Ig expression prolongs recombinant gene expression in skeletal muscle. Molecular Therapy 3: 892–900. Mendell JR, Kissel JT, Amato AA et al. (1995) Myoblast transfer in the treatment of Duchenne’s muscular dystrophy. New England Journal of Medicine 333: 832–838. Moser H (1984) Duchenne muscular dystrophy: pathogenetic aspects and genetic prevention. Human Genetics 66: 17–40. Neuromuscular Disorders. Gene tables for neuromuscular disorders http://www.elsevier.com/homepage/sah/nmd/menu.html Partridge TA (1991) Myoblast transfer: a possible therapy for inherited myopathies? Muscle and Nerve 14: 197–212. Phase 2 clinical trial of PTC124 to promote read-through of stop mutations in DMD http://www.ptcbio.com/ 3.1.1_generic_disorders.aspx Trial of AAV delivery of a microdystrophin expression construct by intramuscular injection in DMD boys http://www.mdausa.org/research/060329dmd_gene_therapy.html Trial of AAV therapy for gamma-sarcoglycanopathy http:// www.genethon.fr/index.php?id=49&L=1 Trial on use of morpholino antisense oligonucleotides to induce skipping of exons such as to restore open reading frame in the mutated dystrophin gene http://www.clinicaltrials.gov/ct/ show/NCT00159250?order=1 Walton J and Gardner-Medwin D (1981) The muscular dystrophies. In: Walton JN (ed.) Disorders of Voluntary Muscle, pp. 481–524. Edinburgh, UK: Churchill Livingstone.


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