Duchenne muscular dystrophy (DMD) is an X-linked, lethal genetic disorder affecting the skeletal muscle compartment, and is caused by mutation(s) in the dystrophin gene. Gene delivery of microdystrophin constructs using adeno-associated virus (AAV) and antisense-mediated exon skipping restoring the genetic reading frame are two of the most promising therapeutic strategies for DMD. Both approaches use microdystrophin proteins either directly as a desired construct for gene delivery, using the capacity-limited AAV vectors, or as the therapeutic outcome of gene splicing. Although functionality of the resulting artificial dystrophin proteins can be predicted in silico, experimental evidence usually obtained in transgenic mice is required before human trials. However, the enormous number of potential constructs makes screening assays for dystrophin protein function in vitro and in vivo highly desirable. Here we present data showing that functionality of microdystrophins can be assessed using relatively simple and fast techniques.
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References
1.
Aartsma-RusA., JansonA.A., HeemskerkJ.A., De WinterC.L., van OmmenG.J., van DeutekomJ.C.2006. Therapeutic modulation of DMD splicing by blocking exonic splicing enhancer sites with antisense oligonucleotides. Ann. N. Y. Acad. Sci., 1082:74–76.
2.
AcsadiG., DicksonG., LoveD.R., JaniA., WalshF.S., GurusingheA., WolffJ.A., DaviesK.E.1991. Human dystrophin expression in mdx mice after intramuscular injection of DNA constructs. Nature, 352:815–818.
3.
AcsadiG., LochmullerH., JaniA., HuardJ., MassieB., PrescottS., SimoneauM., PetrofB.J., KarpatiG.1996. Dystrophin expression in muscles of mdx mice after adenovirus-mediated in vivo gene transfer. Hum. Gene Ther., 7:129–140.
AmannK.J., RenleyB.A., ErvastiJ.M.1998. A cluster of basic repeats in the dystrophin rod domain binds F-actin through an electrostatic interaction. J. Biol. Chem., 273:28419–28423.
6.
AmannK.J., GuoA.W., ErvastiJ.M.1999. Utrophin lacks the rod domain actin binding activity of dystrophin. J. Biol. Chem., 274:35375–35380.
7.
AngeliniC.2007. The role of corticosteroids in muscular dystrophy: A critical appraisal. Muscle Nerve, 36:424–435.
8.
BanksG.B., GregorevicP., AllenJ.M., FinnE.E., ChamberlainJ.S.2007. Functional capacity of dystrophins carrying deletions in the N-terminal actin-binding domain. Hum. Mol. Genet., 16:2105–2113.
9.
BatchelorC.L., WinderS.J.2006. Sparks, signals and shock absorbers: How dystrophin loss causes muscular dystrophy. Trends Cell Biol., 16:198–205.
10.
BeggsA.H., HoffmanE.P., SnyderJ.R., ArahataK., SpechtL., ShapiroF., AngeliniC., SugitaH., KunkelL.M.1991. Exploring the molecular basis for variability among patients with Becker muscular dystrophy: Dystrophin gene and protein studies. Am. J. Hum. Genet., 49:54–67.
11.
BentzingerC.F., BarzaghiP., LinS., RueggM.A.2005. Overexpression of mini-agrin in skeletal muscle increases muscle integrity and regenerative capacity in laminin-α2-deficient mice. FASEB J., 19:934–942.
12.
BönnemannC.G., Passos-BuenoM.R., McNallyE.M., VainzofM., de Sa MoreiraE., MarieS.K., PavanelloR.C., NoguchiS., OzawaE., ZatzM., KunkelL.M.1996. Genomic screening for β-sarcoglycan gene mutations: Missense mutations may cause severe limb-girdle muscular dystrophy type 2E (LGMD 2E)Hum. Mol. Genet., 5:1953–1961.
13.
BrunC., SuterD., PauliC., DunantP., LochmullerH., BurgunderJ.M., SchumperliD., WeisJ.2003. U7 snRNAs induce correction of mutated dystrophin pre-mRNA by exon skipping. Cell Mol. Life Sci., 60:557–566.
ChakkalakalJ.V., ThompsonJ., ParksR.J., JasminB.J.2005. Molecular, cellular, and pharmacological therapies for Duchenne/Becker muscular dystrophies. FASEB J., 19:880–891.
16.
ChakkalakalJ.V., MichelS.A., ChinE.R., MichelR.N., JasminB.J.2006. Targeted inhibition of Ca2+/calmodulin signaling exacerbates the dystrophic phenotype in mdx mouse muscle. Hum. Mol. Genet., 15:1423–1435.
17.
CorradoK., RafaelJ.A., MillsP.L., ColeN.M., FaulknerJ.A., WangK., ChamberlainJ.S.1996. Transgenic mdx mice expressing dystrophin with a deletion in the actin-binding domain display a “mild Becker” phenotype. J. Cell Biol., 134:873–884.
18.
CrawfordG.E., FaulknerJ.A., CrosbieR.H., CampbellK.P., FroehnerS.C., ChamberlainJ.S.2000. Assembly of the dystrophin-associated protein complex does not require the dystrophin COOH-terminal domain. J. Cell Biol., 150:1399–1410.
19.
CrossR.A., StewartM., Kendrick-JonesJ.1990. Structural predictions for the central domain of dystrophin. FEBS Lett., 262:87–92.
20.
DeconinckN., DanB.2007. Pathophysiology of duchenne muscular dystrophy: Current hypotheses. Pediatr. Neurol., 36:1–7.
21.
DongJ.Y., FanP.D., FrizzellR.A.1996. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Hum. Gene Ther., 7:2101–2112.
22.
DubowitzV.1987. A Colour Atlas of Muscle Disorders in Childhood. Mosby International: London.
23.
DudleyR.W.R., LuY., GilbertR., MateckiS., NalbantogluJ., PetrofB.J., KarpatiG.2004. Sustained improvement of muscle function one year after full-length dystrophin gene transfer into mdx mice by a gutted helper-dependent adenoviral vector. Hum. Gene Ther., 15:145–156.
24.
EngelA.G., OzawaE.EngelA.G., Franzini-ArmstrongC.2004. Dystrophinopathies. Myology, 3rd ed.. McGraw-Hill: New York, 961–1025.
25.
EnglandS.B., NicholsonL.V., JohnsonM.A., ForrestS.M., LoveD.R., Zubrzycka-GaarnE.E., BulmanD.E., HarrisJ.B., DaviesK.E.1990. Very mild muscular dystrophy associated with the deletion of 46% of dystrophin. Nature, 343:180–182.
26.
ErvastiJ.M.2007. Dystrophin, its interactions with other proteins, and implications for muscular dystrophy. Biochim. Biophys. Acta, 1772:108–117.
27.
ErvastiJ.M., RybakovaI.N., AmannK.J.1997. A multiple site, side binding model for the interaction of dystrophin with F-actin. Soc. Gen. Physiol. Ser., 52:31–44.
28.
FabbS.A., WellsD.J., SerpenteP., DicksonG.2002. Adeno-associated virus vector gene transfer and sarcolemmal expression of a 144 kDa micro-dystrophin effectively restores the dystrophin-associated protein complex and inhibits myofibre degeneration in nude/mdx mice. Hum. Mol. Genet., 11:733–741.
FriedrichO., von WegnerF., ChamberlainJ.S., FinkR.H., RohrbachP.2008. L-type Ca2+ channel function is linked to dystrophin expression in mammalian muscle. PLoS ONE, 3:e1762.
31.
GilbertR., DudleyR.W.R., LiuA., PetrofB.J., NalbantogluJ., KarpatiG.2003. Prolonged dystrophin expression and functional correction of mdx mouse muscle following gene transfer with a helper-dependent (gutted) adenovirus-encoding murine dystrophin. Hum. Mol. Genet., 12:1287–1299.
32.
GisselH.2005. The role of Ca2+ in muscle cell damage. Ann. N. Y. Acad. Sci., 1066:166–180.
33.
GregorevicP., AllenJ.M., MinamiE., BlankinshipM.J., HaraguchiM., MeuseL., FinnE., AdamsM.E., FroehnerS.C., MurryC.E., ChamberlainJ.S.2006. rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice. Nat. Med., 12:787–789.
34.
GroundsM.D., SorokinL., WhiteJ.2005. Strength at the extracellular matrix–muscle interface. Scand. J. Med. Sci. Sports, 15:381–391.
35.
HamerP.W., McGeachieJ.M., DaviesM.J., GroundsM.D.2002. Evans blue dye as an in vivo marker of myofibre damage: Optimising parameters for detecting initial myofibre membrane permeability. J. Anat., 200:69–79.
36.
HarperS.Q., HauserM.A., DelloRussoC., DuanD., CrawfordR.W., PhelpsS.F., HarperH.A., RobinsonA.S., EngelhardtJ.F., BrooksS.V., ChamberlainJ.S.2002. Modular flexibility of dystrophin: Implications for gene therapy of Duchenne muscular dystrophy. Nat. Med., 8:253–261.
37.
HoffmanE.P., BrownR.H.Jr., KunkelL.M.1987. Dystrophin: The protein product of the Duchenne muscular dystrophy locus. Cell, 51:919–928.
38.
HortonR.M., HuntH.D., HoS.N., PullenJ.K., PeaseL.R.1989. Engineering hybrid genes without the use of restriction enzymes: Gene splicing by overlap extension. Gene, 77:61–68.
39.
KochanekS., ClemensP.R., MitaniK., ChenH.H., ChanS., CaskeyC.T.1996. A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and β-galactosidase. Proc. Natl. Acad. Sci. U.S.A., 93:5731–5736.
40.
LeijendekkerW.J., PassaquinA.C., MetzingerL., RueggU.T.1996. Regulation of cytosolic calcium in skeletal muscle cells of the mdx mouse under conditions of stress. Br. J. Pharmacol., 118:611–616.
41.
LiuM., YueY., HarperS.Q., GrangeR.W., ChamberlainJ.S., DuanD.2005. Adeno-associated virus-mediated microdystrophin expression protects young mdx muscle from contraction-induced injury. Mol. Ther., 11:245–256.
42.
LuQ.L., MorrisG.E., WiltonS.D., LyT., Artem'yevaO.V., StrongP., PartridgeT.A.2000. Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion. J. Cell Biol., 148:985–996.
43.
LuQ.L., RabinowitzA., ChenY.C., YokotaT., YinH., AlterJ., JadoonA., Bou-GhariosG., PartridgeT.2005. Systemic delivery of antisense oligoribonucleotide restores dystrophin expression in body-wide skeletal muscles. Proc. Natl. Acad. Sci. U.S.A., 102:198–203.
44.
MarquezM.J., FerrettiR., VomeroV.U., MinatelE., NetoH.S.2007. Intrinsic laryngeal muscles are spared from myonecrosis in the mdx mouse model of Duchenne muscular dystrophy. Muscle Nerve, 35:349–353.
45.
MirabellaM., GalluzziG., ManfrediG., BertiniE., RicciE., De LeoR., TonaliP., ServideiS.1998. Giant dystrophin deletion associated with congenital cataract and mild muscular dystrophy. Neurology, 51:592–595.
46.
McMahonJ.M., SignoriE., WellsK.E., FazioV.M., WellsD.J.2001. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase: Increased expression with reduced muscle damage. Gene Ther., 16:1264–1270.
47.
MolnarM.J., GilbertR., LuY., LiuA.B., GuoA., LarochelleN., OrloppK., LochmullerH., PetrofB.J., NalbantogluJ., KarpatiG.2004. Factors influencing the efficacy, longevity, and safety of electroporation-assisted plasmid-based gene transfer into mouse muscles. Mol. Ther., 10:447–455.
48.
MorganJ.E., BeauchampJ.R., PagelC.N., PeckhamM., AtaliotisP., JatP.S., NobleM.D., FarmerK., PartridgeT.A.1994. Myogenic cell lines derived from transgenic mice carrying a thermolabile T antigen: A model system for the derivation of tissue-specific and mutation-specific cell lines. Dev. Biol., 162:486–498.
RafaelJ.A., CoxG.A., CorradoK., JungD., CampbellK.P., ChamberlainJ.S.1996. Forced expression of dystrophin deletion constructs reveals structure-function correlations. J. Cell Biol., 134:93–102.
51.
RybakovaI.N., AmannK.J., ErvastiJ.M.1996. A new model for the interaction of dystrophin with F-actin. J. Cell Biol., 135:661–672.
52.
SquireS., RaymackersJ.M., VandebrouckC., PotterA., TinsleyJ., FisherR., GillisJ.M., DaviesK.E.2002. Prevention of pathology in mdx mice by expression of utrophin: Analysis using an inducible transgenic expression system. Hum. Mol. Genet., 11:3333–3344.
53.
TinsleyJ., DeconinckN., FisherR., KahnD., PhelpsS., GillisJ.M., DaviesK.1998. Expression of full-length utrophin prevents muscular dystrophy in mdx mice. Nat. Med., 4:1441–1444.
54.
TinsleyJ.M., PotterA.C., PhelpsS.R., FisherR., TrickettJ.I., DaviesK.E.1996. Amelioration of the dystrophic phenotype of mdx mice using a truncated utrophin transgene. Nature, 384:349–353.
TurkR., SterrenburgE., de MeijerE.J., van OmmenG.J., den DunnenJ.T., ’t HoenP.A.2005. Muscle regeneration in dystrophin-deficient mdx mice studied by gene expression profiling. BMC Genomics, 6:98.
57.
van OmmenG.J., van DeutekomJ., Aartsma-RusA.2008. The therapeutic potential of antisense-mediated exon skipping. Curr. Opin. Mol. Ther., 10:140–149.
58.
WangZ., KuhrC.S., AllenJ.M., BlankinshipM., GregorevicP., ChamberlainJ.S., TapscottS.J., StorbR.2007. Sustained AAV-mediated dystrophin expression in a canine model of Duchenne muscular dystrophy with a brief course of immunosuppression. Mol. Ther., 15:1160–1166.
59.
WarnerL.E., DelloRussoC., CrawfordR.W., RybakovaI.N., PatelJ.R., ErvastiJ.M., ChamberlainJ.S.2002. Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin–glycoprotein complex and partially prevents dystrophy. Hum. Mol. Genet., 11:1095–1105.
60.
WiendlH., HohlfeldR., KieseierB.C.2005. Immunobiology of muscle: Advances in understanding an immunological microenvironment. Trends Immunol., 26:373–380.
61.
WinderS.J., GibsonT.J., Kendrick-JonesJ.1995. Dystrophin and utrophin: The missing links!FEBS Lett., 369:27–33.
62.
XiaoX., LiJ., SamulskiR.J.1996. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector. J. Virol., 70:8098–8108.
63.
YoshimuraM., SakamotoM., IkemotoM., MochizukiY., YuasaK., Miyagoe-SuzukiY., TakedaS.2004. AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype. Mol. Ther., 10:821–828.
64.
YuasaK., SakamotoM., Miyagoe-SuzukiY., TanouchiA., YamamotoH., LiJ., ChamberlainJ.S., XiaoX., TakedaS.2002. Adeno-associated virus vector-mediated gene transfer into dystrophin-deficient skeletal muscles evokes enhanced immune response against the transgene product. Gene Ther., 9:1576–1588.
65.
YuasaK., YoshimuraM., UrasawaN., OhshimaS., HowellJ.M., NakamuraA., HijikataT., Miyagoe-SuzukiY., TakedaS.2007. Injection of a recombinant AAV serotype 2 into canine skeletal muscles evokes strong immune responses against transgene products. Gene Ther., 14:1249–1260.
66.
YueY., LiuM., DuanD.2006. C-terminal-truncated microdystrophin recruits dystrobrevin and syntrophin to the dystrophin-associated glycoprotein complex and reduces muscular dystrophy in symptomatic utrophin/dystrophin double-knockout mice. Mol. Ther., 14:79–87.
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