Expert opinion on biological therapy.
`v. 9, no. 7 (July 2009)
`General Collection
`W1 EX52L
`2009-07-11 12:32:54
`
`Ww\Wa©&l~IDaC3®(il)
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`informa
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`PHARMACEUTICAL SCIENCE
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`

`

`ExP,ert
`Opinion on Biological Therapy
`
`Aims and scope
`Expert Opinion on Biological Therapy is a MEDLINE-indexed, peer-reviewed, international
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`This material may be protected by Copyright law (Title 17 U.S. Code)
`
`ExP.,ert
`Opinion
`
`Introduction
`1.
`2. Affected genes and pathology
`of MD
`3. Conventional gene replacement
`strategies
`4. RNA-based approaches
`5. Gene modification for cell
`transplantation in the ex vivo
`approach
`6. Myostatin blockade to
`manipulate muscle physiology
`7. Expert opinion
`
`informa
`
`healthcare
`
`Review
`
`Gene therapy for muscular
`dystrophy: current progress
`and future prospects
`Capucine Troller, Takis Athanasopoulos, Linda Popplewell, Alberto Malerba &
`George Dickson t
`Royal Hollo wr1y, Eghr1111, Surrey, UK
`
`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.
`
`Keywords: AAV, antisense therapy, clinical trial, gene replacement, gene therapy, lentivirus,
`muscular dystrophy
`
`Expert Opin. Biol. Ther. (2009) 9(7):849-866
`
`1. Introduction
`
`Muscular dystrophies (MD) refers to a heterogeneous group of degenerative
`muscle genetic diseases. Striated muscle tissue accounts for 35% of body mass,
`and has more than 80 associated monogenic pathologies. Among these, the muscular
`dystrophies incorporate a range of severe (often lethal) inherited muscle wasting
`disorders afflicting more than 2 million family members in Europe. Both skeletal
`and cardiac muscles are affected, leading to progressive loss of ambulatory, respiratory
`and cardiac functions . The most frequent muscular dystrophy is Duchenne
`muscular dystrophy (DMD), caused by mutations in the dystrophin gene, and
`affecting I in 3500 male births. Other muscular dystrophies include limb-girdle
`(LGMD), congenital (CMD), facioscapulohumeral (FSHD), myotonic (DMI, DM2),
`oculopharyngeal (OPMD), distal (DD) and Emery-Dreifuss (EDMD) muscular
`dystrophy (Table I). Genes associated with muscular dystrophies encode proteins of
`the plasma membrane, extracellular matrix, sarcomere, Z band, as well as nuclear
`components of the striated muscle cell (Figure 1)11.2] .
`So far there is no corrective therapy for any of the muscular dystrophies. Current
`conventional therapies are limited to supportive care including surgery, corticosteroid
`administration for muscle weakness, medication to counter cardiomyopathy,
`
`10.1517/14712590903029164 © 2009 lnforma UK Ltd ISSN 1471-2598
`All rights reservJIJ:i~~~n'irFw!'lm!Etll in part not permitted
`attihe NLM .a nd m ay be
`Soub,ject USCo;pyright Laws
`
`849
`
`

`

`Gene therapy for muscular dystrophy: current progress and future prospects
`
`Table 1. Gene products affected in the commonest muscular dystrophies.
`
`Disease
`
`Inheritance
`
`Gene product
`
`Localisation
`
`Year
`
`X-linked muscular dystrophy
`
`Duchenne/Becker muscular
`dystrophy (DMD/BMD)
`
`XR
`
`Dystrophin (DMD)
`
`Cytoskeleton
`
`Nucleus
`
`Nucleus
`
`Nucleus
`
`Cytoskeleton
`
`Nucleus
`Sarcolemma
`
`Cytosol
`
`Sarcolemma
`Sarcolemma
`
`Sarcolemma
`
`Sarcolemma
`Sarcolemma
`
`Cytoskeleton
`
`Sarcomere
`
`Emerin (EMD)
`
`Four and a half LIM domain
`1 (FHL1)
`
`Lamin A/C
`
`Lamin A/C
`
`Myotilin (titin immunoglobulin
`domain protein)
`
`Lamin A/C
`Caveolin-3
`
`?
`?
`?
`?
`Calpain-3 (CAPN3)
`
`Dysferlin (DYSF)
`Gamma-sarcoglycan
`
`Alpha-sacroglycan
`
`Beta-sarcoglycan
`Delta-sarcoglycan
`
`Telethonin, titin-cap
`Tripartite motif-containing
`32 (ubiquitin ligase)
`Fukutin related protein (FKRP)
`
`Titin (TTN)
`Protei n-O-ma n nosyl-tr a nsferase
`1 (POMT1)
`
`Anoctarnin 5 (ANOS)
`
`Fukutin (FCMD)
`Protein-O-mannosyl-transferase
`2 (POMT2)
`
`1986
`
`1986
`
`2008
`
`1999
`
`2000
`
`1992
`
`1997
`1998
`
`1999
`1997
`2003
`2005
`
`1991
`
`1994
`1992
`
`1994
`1995
`1996
`
`1997
`
`1998
`
`2000
`
`2003
`
`2005
`
`2007
`
`2006
`
`2007
`
`1998
`
`2002
`
`Emery-Dreifuss muscular
`dystrophy (EDMD) type 1
`Emery-Dreifuss muscular
`dystrophy (EDMD) type 2
`Emery-Dreifuss muscular
`dystrophy (EDMD)
`Emery-Dreifuss muscular
`dystrophy (EDMD)
`Limb-girdle muscular dystrophy (LGMD)
`AD
`
`XR
`
`XR
`
`AD
`
`AR
`
`LGMD 1A
`
`LGMD 1B
`
`LGMD 1C
`LGMD 1D
`LGMD 1 E
`LGMD 1F
`LGMD 1G
`LGMD 2A
`
`LGMD 2B
`LGMD 2C
`LGMD 2D
`LGMD 2E
`LGMD 2F
`
`LGMD 2G
`LGMD 2H
`
`LGMD 21
`
`LGMD 2J
`LGMD 2K
`
`LGMD 2L
`LGMD 2M
`LGMD 2N
`
`Distal muscular dystrophy
`
`AD
`AD
`AD
`
`AD
`AD
`
`AD
`AR
`
`AR
`AR
`AR
`AR
`AR
`AR
`AR
`
`AR
`AR
`AR
`
`AR
`
`AR
`AR
`
`AR
`
`Miyoshi myopathy
`Tibial muscular dystrophy
`
`AD
`Congenital muscular dystrophy (CMD)
`
`Dysferlin (DYSF)
`
`Titin (TTN)
`
`Sarcolernma
`
`Sarcomere
`
`classic
`
`AR
`
`Laminin alpha2 (LAMA2)
`
`Extracellular matrix
`
`1995
`
`Modes of inheritance.
`AR: Autosomal recessive; AD: Autosomal dominant; XR: X-linked recessive; ?: Unknown.
`
`850
`
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`at t he NLM and m ay be
`5'ubject US Copyright Laws
`
`

`

`Table 1. Gene products affected in the commonest muscular dystrophies (continued).
`
`Trollet, Athanasopoulos, Popplewell, Malerba & Dickson
`
`Disease
`
`Fukuyama
`
`CMD and abnormal glycosylation
`of dystroglycan
`
`Walker-Warburg syndrome
`
`Walker-Warburg syndrome
`
`Walker-Warburg syndrome
`
`Other forms of muscular dystrophy
`
`Bethlem myopathy
`
`Epidermolysis bullosa and MD
`
`Oculopharyngeal muscular
`dystrophy (OPMD)
`
`Facioscapulohumeral muscular
`dystrophy (FSHD)
`
`Myotonic dystrophy type 1
`
`Myotonic dystrophy type 2
`
`Inheritance
`
`Gene product
`
`Localisation
`
`AR
`
`AR
`
`AR
`
`AR
`
`AR
`
`AD
`
`AR
`
`AD
`
`AD
`
`AD
`
`AD
`
`Fukutin (FCMD)
`
`Like-glycosyl transferase (LARGE)
`
`Protei n-O-ma n nosy!-transferase
`1 (POMT1)
`
`Protein-O-mannosyl-transferase
`2 (POMT2)
`
`Fukutin related protein (FKRP)
`
`Collagen VI
`
`Plectin (PLEC 1)
`
`Poly(A) binding protein nuclear
`1 (PABPN1)
`
`?
`
`Dystrophia myotonica protein
`kinase (DMPK)
`
`Zinc finger protein 9 (ZNF9)
`
`Extracellular matrix
`
`Cytoskeleton
`
`Nucleus
`
`Modes of inheritance.
`AR: Autosomal recessive; AD: Autosomal dominant; XR: X-linked recessive; ?: Unknown.
`
`Year
`
`1993
`
`2003
`
`2006
`
`2005
`
`2004
`
`1996
`
`1996
`
`1998
`
`1990
`
`1992
`
`2001
`
`-
`
`=-:;.:,~~-~-==:::::==""'~~......_=::::::::==-✓7~c::::::::::::;;;__
`
`Sarcoglycans
`LGMD 2D (a.)
`LGMD 2E (P)
`
`LGMD 2F (S)
`
`Laminin-2
`(CMD)
`..r--,,..,_
`
`Basal membrane proteins
`
`LGMD 2C (y) 600 ·· ....... )
`"-c;----....,.: .... 7 °\ Dystroglycans
`-o:Ol:O:it0J~~
`
`\.,
`Caveolin 3
`(LGMD1C)
`
`Dysferlin
`(LGMD28)
`(MM)
`
`\.,1
`D ys t r~ph in ~1-Jl··.1•··.·.,_·•- S. yntrophins
`~ ,"· {zi,;; P?
`. .
`(DMD/BMD)
`~L.i / ',\:i.. ,,
`~
`Synco1iln
`· .jC" ~:~~~~--
`'-~fi ~ <...;; >-
`Dystrobrevin ' o ·
`%;.. ,t:2; &:
`. ·-....,,
`\ Contractile proteins
`-~ \
`~ '
`j
`....__ ____ . Telethonin (LGMD2G)
`Emerin (EDMD)
`Myotilin (LGMD1A)
`
`Actin filaments
`
`~
`
`(LGMD2A)
`
`@
`
`(CMD)
`(LGMD2)
`
`(
`
`Nuclear membrane
`
`LaminNC
`(LGMD18)
`(EDMD-AD)
`
`8 (OPMD)
`
`Figure 1. Schematic representation of the dystrophin-associated protein complex (DPC) and some other proteins of the
`muscle fibres affected in muscular dystrophies. The corresponding diseases are shown in parentheses.
`AD: Autosomal dominant; BMD: Becker muscular dystrophy; CMD: Congenital muscular dystrophy; DG: Dystroglycan; DMD: Duchenne muscular dystrophy; EDMD:
`Emery-dreifuss muscular dystrophy; FKRP: Fukutin-related protein; LGMD: Limb-girdle muscular dystrophy; MM: Miyoshi myopathy; NOS: Nitric oxide synthase;
`OPMD: Oculopharyngeal muscular dystrophy; PABPN1: Poly(A) binding protein nuclear 1.
`
`Expert Opin. Biol. Ther. (2009) 9(7)
`Th is m a t eria I was cacpied
`at the NLM and m ay be
`'icubject US O:i.,pyTig;ht Laws
`
`851
`
`

`

`..
`
`Y for muscular dystrophy: current progress and future prospects
`h
`Genet erap
`
`c0 r respiratory failure, and physiotherapy. These
`·
`·1
`vent! auon n
`treatments partially alleviate signs and_ symptoms, but do
`not directly target the disease me_chamsm, nor reverse the
`phenotype. With progress made m the knowledge ~f the
`genetics, molecular biology, vectorology and mus~le phys10logy
`of muscular dystrophies, several new strategies based on
`pharmacology or gene therapy are currently being developed
`and hold promise in a near fu_ture. G~ne ~herapy refers to
`the introduction of a therapeutic nucleic acid (DNA, RNA,
`oligonucleotide) into targeted cells in order to_ mod!fy and
`improve the physiology of these cells. _In this review _we
`focus on the main gene therapy strategies currently being
`designed. Some of these strategies could be applied to any
`muscular dystrophy, but the vast majority will be specific of each
`disease. The major challenges presented in the development of
`gene therapy for muscular dystrophies inclu~e: the need to
`target different muscles in the body, and m the case of
`DMD this includes the respiratory muscles/diaphragm and
`heart; optimization of delivery (intram~scular versus intra(cid:173)
`vascular); the need for long-term expression of the transgene;
`the role of potential immune response; problem of fibrosis;
`and prevention of lesions versus
`replacement/repair of
`necrotic fibres.
`
`2. Affected genes and pathology of MD
`
`2.1 Duchenne muscular dystrophy and Becker
`muscular dystrophy
`The X-linked muscle-wasting disease Duchenne muscular
`dystrophy (DMD) is caused by mutations in the gene encoding
`dystrophin. New insights into the pathophysiolob'Y of dystrophic
`muscle, the identification of compensating proteins, and the
`discovery of new binding partners are paving the way for
`novel therapeutic strategies to treat this fatal muscle disease.
`Dystrophin is required for the assembly of the dystrophin(cid:173)
`glycoprotein complex, and provides a mechanically strong link
`between the cytoskeleton and the extracellular matrix. Several
`proteins in the complex also participate in signaling cascades,
`but the relationship between these signaling and mechanical
`functions in the development of muscular dystrophy is
`unclear [I), suggesting that mechanical destabilization, rather
`than signaling dysfunction, is the primary cause of myofibrc
`necrosis in dystrophin-deficicnt muscle.
`Becker muscular dystrophy (13MD)
`is another disease
`caused by mutations of the dystrophin gene. Generally the
`reading-frame hypothesis holds; if the deletion maintains the
`open reading frame of the dystrophin gene, semi-functional,
`internally deleted dystrophin protein is expressed and less
`clinically severe BMD results, whereas the deletions that
`disrupt the reading frame, as seen in DMD, produce severely
`truncated protein that is unstable, and the more severe clinical
`outcome. There is no precise correlation between clinical
`severity of BMD and deletion pattern, but the deletions are
`more homogeneous, while those seen in DMD are more varied
`in position and extent.
`
`2.2 Other muscular dystrophies
`The number of genes associated with autosomal recessive
`limb-girdle muscular dystrophy has been extended to at least
`14 and the phenotypic spectrum has been broadened [3].
`Mutations in at least six genes (protein-0-mannosyl trans(cid:173)
`ferase (POMT) I, POMT2, protein 0-linked man nose p I ,2-
`N-acetylglucosaminyltransferase I (POMGnTI), Fukmin,
`fukutin-related protein (FKRP) and likc-glycosyltransfcrasc
`(LARGE)) have so far been identified in patients with a dystro(cid:173)
`glycanopathy. Allelic mutations in each of these genes can result
`in a wide spectrum of clinical conditions, ranging from severe
`congenital onset with associated structural brain malformations
`(Walker-Warburg syndrome; muscle-eye-brain disease; Fukuyarna
`muscular dystrophy; congenital muscular dystrophy type ID)
`to a relatively milder congenital variant with no brain involve(cid:173)
`ment (congenital muscular dystrophy type IC), and to LGMD
`type 2 variants with onset in childhood or adult life (LGMD2I,
`LGMD2L, and LGMD2N). In particular overexpression of
`LARGE-2 is capable of restoring dystroglycan glycosylation and
`laminin binding properties in primary cell cultures of patients
`affected by different genetically defined dystroglycanopathy
`variants [4]. A functional calpain 3 protease is not mandatory
`for muscle to form in vivo but it is a pre-requisite for muscle
`to remain healthy [5]. Mutations in genes encoding the nuclear
`envelope proteins emerin and lamin A/C lead to a range of
`tissue-specific degenerative diseases. These include dilated
`cardiomyopathy, LGMD and X-linked and autosomal domi(cid:173)
`nant EDMD [6]. EDMD is caused by mutations in either the
`gene encoding lamin NC (LMNA) located at Iq21. 2-q21.
`3 or emerin (EMO) located at Xq28 [7]. It has been 10 years
`since the identification of the poly(A) binding protein
`nuclear I (PABPNJ) gene with a (GCN)(n)/polyalaninc abnor(cid:173)
`mal expansion responsible for OPMD [8]. Expansion of CTG
`triplet repeats in the 3' untranslated region of the dystrophia
`myotonica protein kinase (DMPK') gene causes the autosomal
`dominant myotonic dystrophy type I (DMI) also known as
`Steincrt's disease [9].
`
`3. Conventional gene replacement strategies
`
`Recent years have seen the development of a number of
`strategics and tools for effective gene replacement therapies.
`In this section we will mainly concentrate on DMD as the
`prototype target disease. The helper-dependent adenovirus
`(HDAd) vector system has a cloning capacity of up to 37 kb
`enough to carry the full-length dystrophin [to] or utrophin 1111
`cDNA. However, high and long-term expression of dystrophin
`variants transduced to mature muscle still remains difficult
`due to immunogenicity issues, especially from I st or 2nd
`generation Ad vectors and the low expression of the
`coxsackievirus and adenovirus receptors (CAR) in mature
`muscle [12]. Thus, two main gene replacement strategies have
`progressed: intramuscular administration of plasmid DNA, and
`the utilization of AAV vectors to facilitate the administration
`of full and truncated versions of dystrophin (Figure 2). Systemic
`
`852
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`at t h,e N LM a nd may be
`5'ubject USCopcyright Laws
`
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`Trollet, Athanasopoulos, Popplewell, Malerba & Dickson
`
`c;~~
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`4s-55
`e::1,~rm1m El e imm11m1me11mmmmmmmmmmmITT:JfliJmmmm~1-·:~ .hl°fJ..,,1
`
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`
`Actin
`
`Ex
`
`'--=:;::?
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`
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`
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`
`• • • • •
`
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`
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`
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`
`• - - - - • • • • • • • • • • • • -
`
`•
`Mini-dystrophin t.e45 - 55 s17 - 48
`c:c:::,
`__
`Actin r,T!""'1 n
`~~~TIWi a a m~~mrammITT.i~~
`~~nanmmmm~mmmmm~. , ~
`t.H2-R15/t.18-19 C;:)
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`
`..
`t.H2-R15 ~ .
`
`. NOS?
`
`C::::,
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`
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`
`Micro-dystrophins
`c:::r:::::,
`t.DysM3
`r,-=,:,
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`~~ 1."o'.,IW lilt I".•.!...___.,____.~
`
`t.DysCSI
`
`63
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`
`t.3788
`
`~ l!!llll fll llil ~ E!l Bl Ell Bl Bl tm..::.....::
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`~
`
`~H2-R15/t.18-23/t.C
`
`C:::>
`0::::::,
`~lml!lf.liIHiBlml ~-- -·:
`
`Figure 2. Strucure offull-length dystrophin, quasi-dystrophin, mini-dystrophin and micro-dystrophin genes that have been (or are
`currently in progress to be) assessed for the restoration of dystrophic muscle function via potential gene augmentation strategies
`are presented. Full-length dystrophin cDNA can be delivered via plasmid, mini-circles or gutted adenoviral vectors, but further developments/
`technologies covering 'full body' widespread delivery are feasible to arise in the near future . Quasidystrophin, minidystrophin (Becker) gene
`and derivatives were developed according to various truncations and patient data deletions/mutations in dystrophin genes associated with
`mild dystrophinopathy, for example, Becker muscular dystrophy (BMD). These genes can be currently delivered via dual transplicing and/or
`overlapping adeno-associated virus (AA V) or lentivector approaches Microdystrophin genes are much smaller transgenes that were generated
`to determine the minimum requirements for normal dystrophin function and are suitable to be packaged in rAAV or lenti-vectors. We have
`observed previously that semi-functional microdystrophin genes have been constructed with as few as four spectrin-like repeats and two
`to three hinge regions and quite successful microdystrophin constructs in ameliorating Duchenne muscular dystrophy pathology shared a
`common three rod domain hinge spacing (e.g., H 1, H3 and H4) and the presence of eight fully phased spectrin-like repeats. However, under
`the prism of new developments, fully retaining at least three crucial domains in the truncated dystrophin structure, including for example
`the actin-binding site (ABS), nNOS and Dystrophin-associated Protein Complex/syntrophin/dystrobrevin (DPC/syn/dtn) domains is critical.
`*indicates paradigms of full-length, mini or microdystrophin structures where the majority of critical elements/domains are preserved, thus potentially resulting in
`favourite functionally structures.
`C: Carboxy-terminus; CR: Cysteine-rich reg ion; N: amino-terminus.
`
`delivery of plasmid vectors is usually hampered by a lack of
`long-term expression of the transgene, usually one of the
`main goals of gene therapy. In the field of rAAV-mediated
`gene transfer approaches for DMD a range of novel AAV
`scrotypes have been recently been developed 113-15]. However,
`dystrophin gene replacement by a micro-dystrophin or a
`mini-dystrophin alone may not be enough as a therapy, since
`components of the dystrophin-associated protein complex
`
`(DPC, Figure I) and other factors may play a synergistic role
`in the development (and amelioration) of a DMD pathology.
`For instance, absence of nNOS from the sarcolemma can
`impede NO-mediated regulation of vasodilation. Lai et al.
`showed that sarcolemmal targeting of nNOS was dependent
`on the spectrin-like repeats 16 and 17 (Rl 6/17) within the rod
`domain of the dystrophin protein. It has been established
`that the Golgi complex distribution abnormalities seen in mdx
`
`Expert Opin. Biol. Ther. (2009) 9(7)
`Th is mater ia I was co pied
`3ttihe NLM and ma y b,e
`Subject US Cop yright Laws
`
`853
`
`

`

`Gene therapy for muscular dystrophy: current progress and future prospects
`
`skeletal muscle can be rescued by microdystrophin expression [16].
`Overexpression of P-1,4-N-acetyl-galactosaminyl transferase
`2 (Galgt2) in the skeletal muscles of transgenic mice inhibits
`the development of muscular dystrophy in mdx mice [171, ?r
`the use of rMV to deliver the TNF-a soluble receptor, m
`conjunction with microdystrophin, may be of benefit in more
`advanced cases of DMD. Gene transfer approaches are limited
`by difficulties associated with producing su~cient quanti_ties
`of vector, cellular tropism of the vectors, ectopic gene express10n,
`the need for methods to achieve 'whole body' delivery of the
`vector, and inherent immunological challenges [18].
`Non-viral methods of gene delivery and repair provide an
`alternative to viral-based methods. Major concerns with
`nonviral approaches include the effective distribution and
`integration of therapeutic molecules throughout the muscle.
`However, a number of methods have been developed to
`overcome these obstacles. These include electroporation (EP),
`mini-circle development, scaffold/matrix attachment region
`(SIMAR) vectors, use of muscle-specific promoters, delivery
`channels like vascular injection and occlusion, myotoxin(cid:173)
`induced muscle regeneration, and the use of pharmacological
`agents. Intramuscular injection of plasmid is a potential
`alternative to viral vectors for the transfer of therapeutic genes
`into skeletal muscle fibers. Properly optimized EP-assisted
`plasmid-based gene transfer is a feasible, efficient and safe
`method [19] of gene replacement therapy for dystrophin
`deficiency of muscle, but readministration may be necessary [20].
`In addition to the well-defined viral strategies, plasmid vectors
`and the upregulation of utrophin have therapeutic potential [21].
`Utrophin is a ubiquitously expressed homolog of dystrophin
`that is able to perform similar functions, and most importantly
`perhaps is a binding member of the dystroglycan-associated
`protein complex (DAPC). The intravascular delivery of naked
`plasmid DNA (pDNA) to muscle cells is also attractive,
`particularly since many muscle groups would have to be
`targeted for intrinsic muscle disorders such as Duchenne
`muscular dystrophy. High levels of gene expression were first
`achieved by the rapid injection of naked DNA in large volumes
`via an artery with both blood inflow and outflow blocked
`surgically [22]. Intravenous routes have also been shown to be
`effective [23]. For limb muscles, the ability to use a peripheral
`limb vein for injection and a proximal, external tourniquet to
`block blood flow renders the procedure clinically viable [2/4].
`In genetic therapies, the large size of the dystrophin gene
`has necessitated the development and use of novel functional
`minidystrophin and microdystrophin genes, muscle-specific
`promoter systems, and gutted adenoviral systems. Functional
`capacity of a minidystrophin (minidysGFP) and a micrody(cid:173)
`strophin (microdystrophin(AR.4-R.23)) transgene on the mat(cid:173)
`uration and maintenance of neuromuscular junctions (NMJ)
`in mdx mice has been previously tested. MinidysGFP prevented
`fragmentation and the loss of postsynaptic folds at the NMJ.
`In contrast, microdystrophin (AR.4-R.23) was unable to prevent
`synapse fragmentation in the limb muscles despite preventing
`muscle degeneration [25].
`
`Gene replacement using modified viral vectors and plasmids
`has yielded the greatest levels of therapeutic protein expression
`in muscle to date, however there are issues with immune(cid:173)
`mediated responses to the transgene and viral proteins, hin(cid:173)
`dering sustained expression. New efforts have been made to
`make viral delivery of genes safer and sustainable including
`development of hybrid vectors and 'gutted' helper-dependant
`vectors, but one of the most prominent approaches is using
`recombinant adeno-associated viruses (rMV) allowing wide(cid:173)
`spread systemic delivery (as pioneered by Jeff Chamberlain's
`group using AAV6 serotype vectors in 2004 and followed
`shortly by Xiao Xiao's group using AAVS serotype vectors in
`2005) and robust delivery to the heart in certain preclinical
`models. lntravascular administration of recombinant adeno(cid:173)
`associated viral (rMV) vectors carrying a microdystrophin gene
`restores expression of dystrophin in the respiratory, cardiac
`and limb musculature of these mice, considerably reducing
`skeletal muscle pathology and extending lifespan [26].
`Systemic gene delivery of microdystrophin can restore
`ventricular distensibility and protect the mdx myocardium from
`pump dysfunction during adrenergic s