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• W2040287583 abstract "Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology. Limb-girdle muscular dystrophy 2I (LGMD2I) is caused by mutations in the fukutin-related protein (FKRP) gene. Unlike its severe allelic forms, LGMD2I usually involves slower onset and milder course without defects in the central nervous system. The lack of viable animal models that closely recapitulate LGMD2I clinical phenotypes led us to use RNA interference technology to knock down FKRP expression via postnatal gene delivery so as to circumvent embryonic lethality. Specifically, an adeno-associated viral vector was used to deliver short hairpin (shRNA) genes to healthy ICR mice. Adeno-associated viral vectors expressing a single shRNA or two different shRNAs were injected one time into the hind limb muscles. We showed that FKRP expression at 10 months postinjection was reduced by about 50% with a single shRNA and by 75% with the dual shRNA cassette. Dual-cassette injection also reduced a-dystroglycan glycosylation and its affinity to laminin by up to 70% and induced α-dystrophic pathology, including fibrosis and central nucleation, in more than 50% of the myofibers at 10 months after injection. These results suggest that the reduction of approximately or more than 75% of the normal level of FKRP expression induces chronic dystrophic phenotypes in skeletal muscles. Furthermore, the restoration of about 25% of the normal FKRP level could be sufficient for LGMD2I therapy to correct the genetic deficiency effectively and prevent dystrophic pathology. Limb-girdle muscular dystrophies (LGMD) are a group of clinically and genetically heterogeneous muscular diseases that have both autosomal dominant (type 1) and autosomal recessive (type 2) inheritance. The disorders are generally characterized by progressive muscle wasting and weakness of the shoulder and pelvic girdles and often are associated with a wide range of clinical severity.1Bushby K.M. The limb-girdle muscular dystrophies: multiple genes, multiple mechanisms.Hum Mol Genet. 1999; 8: 1875-1882Crossref PubMed Scopus (125) Google Scholar, 2Moreira E.S. Vainzof M. Marie S.K. Sertié A.L. Zatz M. Passos-Bueno M.R. The seventh form of autosomal recessive limb-girdle muscular dystrophy is mapped to 17q11-12.Am J Hum Genet. 1997; 61: 151-159Abstract Full Text PDF PubMed Scopus (115) Google Scholar, 3Bushby K.M. Making sense of the limb girdle muscular dystrophies.Brain. 1999; 122: 1403-1420Crossref PubMed Scopus (152) Google Scholar, 4Guglieri M. Straub V. Bushby K. Lochmuller H. Limb-girdle muscular dystrophies.Curr Opin Neurol. 2008; 21: 576-584Crossref PubMed Scopus (96) Google Scholar, 5Laval S.H. Bushby K.M. Limb-girdle muscular dystrophies: from genetics to molecular pathology.Neuropathol Appl Neurobiol. 2004; 30: 91-105Crossref PubMed Scopus (121) Google Scholar To date, at least 13 subtypes (A–M) of LGMD type 2 have been reported, and the causative genes for each subtype have also been identified; LGMD2I (OMIM_607155) is one of the subsets and is caused by mutations in the gene encoding fukutin-related protein (FKRP). The disease is also one of the more common types of LGMD in Denmark,6Sveen M.L. Schwartz M. Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark.Ann Neurol. 2006; 59: 808-815Crossref PubMed Scopus (120) Google Scholar the United Kingdom,7Poppe M. Cree L. Bourke J. Eagle M. Anderson L.V. Birchall D. Brockington M. Buddles M. Busby M. Muntoni F. Wills A. Bushby K. The phenotype of limb-girdle muscular dystrophy type 2I.Neurology. 2003; 60: 1246-1251Crossref PubMed Scopus (174) Google Scholar Brazil,8Frosk P. Greenberg C.R. Tennese A.A. Lamont R. Nylen E. Hirst C. Frappier D. Roslin N.M. Zaik M. Bushby K. Straub V. Zatz M. de Paula F. Morgan K. Fujiwara T.M. Wrogemann K. The most common mutation in FKRP causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only once and is present in Hutterites and other populations.Hum Mutat. 2005; 25: 38-44Crossref PubMed Scopus (64) Google Scholar and the United States.9Moore S.A. Shilling C.J. Westra S. Wall C. Wicklund M.P. Stolle C. Brown C.A. Michele D.E. Piccolo F. Winder T.L. Stence A. Barresi R. King N. King W. Florence J. Campbell K.P. Fenichel G.M. Stedman H.H. Kissel J.T. Griggs R.C. Pandya S. Mathews K.D. Pestronk A. Serrano C. Darvish D. Mendell J.R. Limb-girdle muscular dystrophy in the United States.J Neuropathol Exp Neurol. 2006; 65: 995-1003Crossref PubMed Scopus (135) Google Scholar, 10Kang P.B. Feener C.A. Estrella E. Thorne M. White A.J. Darras B.T. Amato A.A. Kunkel L.M. LGMD2I in a North American population.BMC Musculoskelet Disord. 2007; 8: 115Crossref PubMed Scopus (26) Google Scholar The onset of LGMD2I can occur from early childhood to adulthood. In addition, cardiac involvement has been frequently reported in patients with LGMD2I.6Sveen M.L. Schwartz M. Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark.Ann Neurol. 2006; 59: 808-815Crossref PubMed Scopus (120) Google Scholar, 7Poppe M. Cree L. Bourke J. Eagle M. Anderson L.V. Birchall D. Brockington M. Buddles M. Busby M. Muntoni F. Wills A. Bushby K. The phenotype of limb-girdle muscular dystrophy type 2I.Neurology. 2003; 60: 1246-1251Crossref PubMed Scopus (174) Google Scholar, 11Gaul C. Deschauer M. Tempelmann C. Vielhaber S. Klein H.U. Heinze H.J. Zierz S. Grothues F. Cardiac involvement in limb-girdle muscular dystrophy 2I: conventional cardiac diagnostic and cardiovascular magnetic resonance.J Neurol. 2006; 253: 1317-1322Crossref PubMed Scopus (38) Google Scholar, 12Poppe M. Bourke J. Eagle M. Frosk P. Wrogemann K. Greenberg C. Muntoni F. Voit T. Straub V. Hilton-Jones D. Shirodaria C. Bushby K. Cardiac and respiratory failure in limb-girdle muscular dystrophy 2I.Ann Neurol. 2004; 56: 738-741Crossref PubMed Scopus (89) Google Scholar, 13Boito C.A. Melacini P. Vianello A. Prandini P. Gavassini B.F. Bagattin A. Siciliano G. Angelini C. Pegoraro E. Clinical and molecular characterization of patients with limb-girdle muscular dystrophy type 2I.Arch Neurol. 2005; 62: 1894-1899Crossref PubMed Scopus (72) Google Scholar By far, the most common mutation in the FKRP gene is the point mutation C826A in the coding sequence, which results in an amino acid change from leucine to isoleucine (L276I) at position 276.6Sveen M.L. Schwartz M. Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark.Ann Neurol. 2006; 59: 808-815Crossref PubMed Scopus (120) Google Scholar, 7Poppe M. Cree L. Bourke J. Eagle M. Anderson L.V. Birchall D. Brockington M. Buddles M. Busby M. Muntoni F. Wills A. Bushby K. The phenotype of limb-girdle muscular dystrophy type 2I.Neurology. 2003; 60: 1246-1251Crossref PubMed Scopus (174) Google Scholar, 8Frosk P. Greenberg C.R. Tennese A.A. Lamont R. Nylen E. Hirst C. Frappier D. Roslin N.M. Zaik M. Bushby K. Straub V. Zatz M. de Paula F. Morgan K. Fujiwara T.M. Wrogemann K. The most common mutation in FKRP causing limb girdle muscular dystrophy type 2I (LGMD2I) may have occurred only once and is present in Hutterites and other populations.Hum Mutat. 2005; 25: 38-44Crossref PubMed Scopus (64) Google Scholar, 9Moore S.A. Shilling C.J. Westra S. Wall C. Wicklund M.P. Stolle C. Brown C.A. Michele D.E. Piccolo F. Winder T.L. Stence A. Barresi R. King N. King W. Florence J. Campbell K.P. Fenichel G.M. Stedman H.H. Kissel J.T. Griggs R.C. Pandya S. Mathews K.D. Pestronk A. Serrano C. Darvish D. Mendell J.R. Limb-girdle muscular dystrophy in the United States.J Neuropathol Exp Neurol. 2006; 65: 995-1003Crossref PubMed Scopus (135) Google Scholar, 10Kang P.B. Feener C.A. Estrella E. Thorne M. White A.J. Darras B.T. Amato A.A. Kunkel L.M. LGMD2I in a North American population.BMC Musculoskelet Disord. 2007; 8: 115Crossref PubMed Scopus (26) Google Scholar Several studies have reported that homozygous L276I mutation is generally associated with a mild phenotype, whereas compound heterozygous mutation tends to produce a more severe course.6Sveen M.L. Schwartz M. Vissing J. High prevalence and phenotype-genotype correlations of limb girdle muscular dystrophy type 2I in Denmark.Ann Neurol. 2006; 59: 808-815Crossref PubMed Scopus (120) Google Scholar, 14Mercuri E. Brockington M. Straub V. Quijano-Roy S. Yuva Y. Herrmann R. Brown S.C. Torelli S. Dubowitz V. Blake D.J. Romero N.B. Estournet B. Sewry C.A. Guicheney P. Voit T. Muntoni F. Phenotypic spectrum associated with mutations in the fukutin-related protein gene.Ann Neurol. 2003; 53: 537-542Crossref PubMed Scopus (199) Google Scholar, 15Walter M.C. Petersen J.A. Stucka R. Fischer D. Schroder R. Vorgerd M. Schroers A. Schreiber H. Hanemann C.O. Knirsch U. Rosenbohm A. Huebner A. Barisic N. Horvath R. Komoly S. Reilich P. Muller-Felber W. Pongratz D. Muller J.S. Auerswald E.A. Lochmuller H. FKRP (826C>A) frequently causes limb-girdle muscular dystrophy in German patients.J Med Genet. 2004; 41: e50Crossref PubMed Scopus (80) Google Scholar, 16Brockington M. Yuva Y. Prandini P. Brown S.C. Torelli S. Benson M.A. Herrmann R. Anderson L.V. Bashir R. Burgunder J.M. Fallet S. Romero N. Fardeau M. Straub V. Storey G. Pollitt C. Richard I. Sewry C.A. Bushby K. Voit T. Blake D.J. Muntoni F. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.Hum Mol Genet. 2001; 10: 2851-2859Crossref PubMed Google Scholar Currently, the diagnosis for LGMD2I is based mainly on clinical evaluations and immunohistochemical analyses of muscle biopsies followed by genetic screening for the FKRP gene,17Evans V. Foster H. Graham I.R. Foster K. Athanasopoulos T. Simons J.P. Dickson G. Owen J.S. Human apolipoprotein E expression from mouse skeletal muscle by electrotransfer of nonviral DNA (plasmid) and pseudotyped recombinant adeno-associated virus (AAV2/7).Hum Gene Ther. 2008; 19: 569-578Crossref PubMed Scopus (15) Google Scholar muscle magnetic resonance imaging, 18Fischer D. Walter M.C. Kesper K. Petersen J.A. Aurino S. Nigro V. Kubisch C. Meindl T. Lochmuller H. Wilhelm K. Urbach H. Schroder R. Diagnostic value of muscle MRI in differentiating LGMD2I from other LGMDs.J Neurol. 2005; 252: 538-547Crossref PubMed Scopus (115) Google Scholar and cardiovascular magnetic resonance imaging.11Gaul C. Deschauer M. Tempelmann C. Vielhaber S. Klein H.U. Heinze H.J. Zierz S. Grothues F. Cardiac involvement in limb-girdle muscular dystrophy 2I: conventional cardiac diagnostic and cardiovascular magnetic resonance.J Neurol. 2006; 253: 1317-1322Crossref PubMed Scopus (38) Google Scholar The human FKRP gene is mapped to chromosome 19q13.3 and consists of four exons,19Driss A. Amouri R. Ben Hamida C. Souilem S. Gouider-Khouja N. Ben Hamida M. Hentati F. A new locus for autosomal recessive limb-girdle muscular dystrophy in a large consanguineous Tunisian family maps to chromosome 19q13.3.Neuromuscul Disord. 2000; 10: 240-246Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar with exon 4 being the single coding exon. The FKRP transcript is expressed predominantly in the skeletal muscle, placenta, and heart.20Brockington M. Blake D.J. Prandini P. Brown S.C. Torelli S. Benson M.A. Ponting C.P. Estournet B. Romero N.B. Mercuri E. Voit T. Sewry C.A. Guicheney P. Muntoni F. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.Am J Hum Genet. 2001; 69: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar The FKRP protein has been shown to localize to the Golgi apparatus,21Esapa C.T. Benson M.A. Schroder J.E. Martin-Rendon E. Brockington M. Brown S.C. Muntoni F. Kroger S. Blake D.J. Functional requirements for fukutin-related protein in the Golgi apparatus.Hum Mol Genet. 2002; 11: 3319-3331Crossref PubMed Scopus (114) Google Scholar, 22Dolatshad N.F. Brockington M. Torelli S. Skordis L. Wever U. Wells D.J. Muntoni F. Brown S.C. Mutated fukutin-related protein (FKRP) localizes as wild type in differentiated muscle cells.Exp Cell Res. 2005; 309: 370-378Crossref PubMed Scopus (17) Google Scholar, 23Keramaris-Vrantsis E. Lu P.J. Doran T. Zillmer A. Ashar J. Esapa C.T. Benson M.A. Blake D.J. Rosenfeld J. Lu Q.L. Fukutin-related protein localizes to the Golgi apparatus and mutations lead to mislocalization in muscle in vivo.Muscle Nerve. 2007; 36: 455-465Crossref PubMed Scopus (25) Google Scholar, 24Torelli S. Brown S.C. Brockington M. Dolatshad N.F. Jimenez C. Skordis L. Feng L.H. Merlini L. Jones D.H. Romero N. Wewer U. Voit T. Sewry C.A. Noguchi S. Nishino I. Muntoni F. Sub-cellular localization of fukutin-related protein in different cell lines and in the muscle of patients with MDC1C and LGMD2I.Neuromuscul Disord. 2005; 15: 836-843Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar but other studies have reported its localization to the endoplasmic reticulum25Matsumoto H. Noguchi S. Sugie K. Ogawa M. Murayama K. Hayashi Y.K. Nishino I. Subcellular localization of fukutin and fukutin-related protein in muscle cells.J Biochem. 2004; 135: 709-712Crossref PubMed Scopus (42) Google Scholar and sarcolemma.26Beedle A.M. Nienaber P.M. Campbell K.P. Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex.J Biol Chem. 2007; 282: 16713-16717Crossref PubMed Scopus (36) Google Scholar Although the precise function of FKRP is not clearly understood, evidence strongly suggests that the protein is involved in post-translational modification of Eα-dystroglycan,16Brockington M. Yuva Y. Prandini P. Brown S.C. Torelli S. Benson M.A. Herrmann R. Anderson L.V. Bashir R. Burgunder J.M. Fallet S. Romero N. Fardeau M. Straub V. Storey G. Pollitt C. Richard I. Sewry C.A. Bushby K. Voit T. Blake D.J. Muntoni F. Mutations in the fukutin-related protein gene (FKRP) identify limb girdle muscular dystrophy 2I as a milder allelic variant of congenital muscular dystrophy MDC1C.Hum Mol Genet. 2001; 10: 2851-2859Crossref PubMed Google Scholar, 26Beedle A.M. Nienaber P.M. Campbell K.P. Fukutin-related protein associates with the sarcolemmal dystrophin-glycoprotein complex.J Biol Chem. 2007; 282: 16713-16717Crossref PubMed Scopus (36) Google Scholar, 27Esapa C.T. McIlhinney R.A. Blake D.J. Fukutin-related protein mutations that cause congenital muscular dystrophy result in ER-retention of the mutant protein in cultured cells.Hum Mol Genet. 2005; 14: 295-305Crossref PubMed Scopus (48) Google Scholar, 28Aravind L. Koonin E.V. The fukutin protein family; predicted enzymes modifying cell-surface molecules.Curr Biol. 1999; 9: R836-R837Abstract Full Text PDF PubMed Google Scholar a critical component of the dystrophin-glycoprotein complex at the sarcolemma.29Ervasti J.M. Campbell K.P. Membrane organization of the dystrophin-glycoprotein complex.Cell. 1991; 66: 1121-1131Abstract Full Text PDF PubMed Scopus (1112) Google Scholar For example, mutations in the FKRP gene are often associated with secondary abnormal glycosylation of α-dystroglycan (hypoglycosylation)20Brockington M. Blake D.J. Prandini P. Brown S.C. Torelli S. Benson M.A. Ponting C.P. Estournet B. Romero N.B. Mercuri E. Voit T. Sewry C.A. Guicheney P. Muntoni F. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.Am J Hum Genet. 2001; 69: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 30Mercuri E. Messina S. Bruno C. Mora M. Pegoraro E. Comi G.P. D'Amico A. Aiello C. Biancheri R. Berardinelli A. Boffi P. Cassandrini D. Laverda A. Moggio M. Morandi L. Moroni I. Pane M. Pezzani R. Pichiecchio A. Pini A. Minetti C. Mongini T. Mottarelli E. Ricci E. Ruggieri A. Saredi S. Scuderi C. Tessa A. Toscano A. Tortorella G. Trevisan C.P. Uggetti C. Vasco G. Santorelli F.M. Bertini E. Congenital muscular dystrophies with defective glycosylation of dystroglycan: a population study.Neurology. 2009; 72: 1802-1809Crossref PubMed Scopus (148) Google Scholar, 31Yamamoto L.U. Velloso F.J. Lima B.L. Fogaca L.L. de Paula F. Vieira N.M. Zatz M. Vainzof M. Muscle protein alterations in LGMD2I patients with different mutations in the fukutin-related protein gene.J Histochem Cytochem. 2008; 56: 995-1001Crossref PubMed Scopus (17) Google Scholar, 32Jimenez-Mallebrera C. Torelli S. Feng L. Kim J. Godfrey C. Clement E. Mein R. Abbs S. Brown S.C. Campbell K.P. Kroger S. Talim B. Topaloglu H. Quinlivan R. Roper H. Childs A.M. Kinali M. Sewry C.A. Muntoni F. A comparative study of alpha-dystroglycan glycosylation in dystroglycanopathies suggests that the hypoglycosylation of alpha-dystroglycan does not consistently correlate with clinical severity.Brain Pathol. 2009; 19: 596-611Crossref PubMed Scopus (90) Google Scholar, 33Brown S.C. Torelli S. Brockington M. Yuva Y. Jimenez C. Feng L. Anderson L. Ugo I. Kroger S. Bushby K. Voit T. Sewry C. Muntoni F. Abnormalities in alpha-dystroglycan expression in MDC1C and LGMD2I muscular dystrophies.Am J Pathol. 2004; 164: 727-737Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar and can cause more severe types of muscular dystrophies, including Walker-Warburg syndrome, muscle-eye-brain disease,34Beltran-Valero de Bernabe D. Voit T. Longman C. Steinbrecher A. Straub V. Yuva Y. Herrmann R. Sperner J. Korenke C. Diesen C. Dobyns W.B. Brunner H.G. van Bokhoven H. Brockington M. Muntoni F. Mutations in the FKRP gene can cause muscle-eye-brain disease and Walker-Warburg syndrome.J Med Genet. 2004; 41: e61Crossref PubMed Scopus (212) Google Scholar and congenital muscular dystrophy type 1C.20Brockington M. Blake D.J. Prandini P. Brown S.C. Torelli S. Benson M.A. Ponting C.P. Estournet B. Romero N.B. Mercuri E. Voit T. Sewry C.A. Guicheney P. Muntoni F. Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin alpha2 deficiency and abnormal glycosylation of alpha-dystroglycan.Am J Hum Genet. 2001; 69: 1198-1209Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar, 35Louhichi N. Triki C. Quijano-Roy S. Richard P. Makri S. Meziou M. Estournet B. Mrad S. Romero N.B. Ayadi H. Guicheney P. Fakhfakh F. New FKRP mutations causing congenital muscular dystrophy associated with mental retardation and central nervous system abnormalities: identification of a founder mutation in Tunisian families.Neurogenetics. 2004; 5: 27-34Crossref PubMed Scopus (71) Google Scholar Although recent clinical studies have made rapid progress in understanding LGMD2I, the lack of a viable animal model for LGMD2I has impeded research into its pathobiology and the development of therapeutics. Targeted deletion of the mouse FKRP gene was embryonically lethal, indicating that FKRP is required for embryo development (unpublished results, Q.L.L.). In humans, no patient has ever been reported to carry homozygous null mutations of the FKRP gene until recently. Dr. van Reeuwijk and colleagues reported that two siblings carrying a homozygous mutation (c.1 A>G, Met1Val) in the start codon of FKRP resulted in Walker-Warburg syndrome, the most severe disorder in the disease spectrum of dystroglycanopathies.36van Reeuwijk J. Olderode-Berends M.J. van den Elzen C. Brouwer O.F. Roscioli T. van Pampus M.G. Scheffer H. Brunner H.G. van Bokhoven H. Hol F.A. A homozygous FKRP start codon mutation is associated with Walker-Warburg syndrome, the severe end of the clinical spectrum.Clin Genet. 2010; 78: 275-281Crossref PubMed Scopus (27) Google Scholar This is highly likely to be a homozygous null FKRP mutation. On the other hand, experiments in our own laboratory and others showed that mice engineered homozygous for the mild L276I missense mutation in the FKRP gene exhibited no appreciable phenotypes (unpublished observations). Recently, Ackroyd and colleagues37Ackroyd M.R. Skordis L. Kaluarachchi M. Godwin J. Prior S. Fidanboylu M. Piercy R.J. Muntoni F. Brown S.C. Reduced expression of fukutin-related protein in mice results in a model for fukutin-related protein associated muscular dystrophies.Brain. 2009; 132: 439-451Crossref PubMed Scopus (57) Google Scholar reported that knockin mice carrying a disease-causing Y307N mutation in the FKRP gene also failed to generate discernible phenotypes. It is interesting to note that if the neomycin-resistant cassette was kept in the mutant FKRP allele, the homozygous Y307N neo mice developed very severe phenotypes, including abnormalities in muscle, eye, and brain, and died immediately after birth. In addition, the levels of FKRP transcripts and α-dystroglycan glycosylation (α-DG) were reduced. These results suggest that a knockdown strategy might be a good alternative for investigating FKRP functions. In support of this idea, it has been demonstrated that down-regulation of FKRP has led to developmental abnormalities in zebrafish morphant embryos.38Thornhill P. Bassett D. Lochmuller H. Bushby K. Straub V. Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of fukutin-related protein (FKRP).Brain. 2008; 131: 1551-1561Crossref PubMed Scopus (57) Google Scholar, 39Kawahara G. Guyon J.R. Nakamura Y. Kunkel L.M. Zebrafish models for human FKRP muscular dystrophies.Hum Mol Genet. 2010; 19: 623-633Crossref PubMed Scopus (62) Google Scholar There is still a need for the generation of a viable LGMD2I mouse model that will recapitulate the clinical phenotypes seen in the majority of LGMD2I patients without brain involvement. In an effort to circumvent the problem of embryonic and neonatal lethality, we adopted RNA interference (RNAi) technology to postnatally knock down FKRP gene expression. Specifically, we used adeno-associated virus (AAV) as an efficient and long-term gene delivery vehicle40Xiao X. Li J. Samulski R.J. Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.J Virol. 1996; 70: 8098-8108Crossref PubMed Google Scholar, 41Kessler P.D. Podsakoff G.M. Chen X. McQuiston S.A. Colosi P.C. Matelis L.A. Kurtzman G.J. Byrne B.J. Gene delivery to skeletal muscle results in sustained expression and systemic delivery of a therapeutic protein.Proc Natl Acad Sci U S A. 1996; 93: 14082-14087Crossref PubMed Scopus (534) Google Scholar, 42Wang B. Li J. Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model.Proc Natl Acad Sci USA. 2000; 97: 13714-13719Crossref PubMed Scopus (405) Google Scholar, 43Xiao X. Li J. Tsao Y.P. Dressman D. Hoffman E.P. Watchko JF Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy.J Virol. 2000; 74: 1436-1442Crossref PubMed Scopus (87) Google Scholar, 44Greelish J.P. Su L.T. Lankford E.B. Burkman J.M. Chen H. Konig S.K. Mercier I.M. Desjardins P.R. Mitchell M.A. Zheng X.G. Leferovich J. Gao G.P. Balice-Gordon R.J. Wilson J.M. Stedman H.H. Stable restoration of the sarcoglycan complex in dystrophic muscle perfused with histamine and a recombinant adeno-associated viral vector.Nat Med. 1999; 5: 439-443Crossref PubMed Scopus (197) Google Scholar, 45Cordier L. Hack A.A. Scott M.O. Barton-Davis E.R. Gao G. Wilson J.M. McNally E.M. Sweeney H.L. Rescue of skeletal muscles of gamma-sarcoglycan-deficient mice with adeno-associated virus-mediated gene transfer.Mol Ther. 2000; 1: 119-129Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 46Dressman D. Araishi K. Imamura M. Sasaoka T. Liu L.A. Engvall E. Hoffman E.P. Delivery of alpha- and beta-sarcoglycan by recombinant adeno-associated virus: efficient rescue of muscle, but differential toxicity.Hum Gene Ther. 2002; 13: 1631-1646Crossref PubMed Scopus (49) Google Scholar, 47High K.A. Clinical gene transfer studies for hemophilia B.Semin Thromb Hemost. 2004; 30: 257-267Crossref PubMed Scopus (65) Google Scholar, 48Mejat A. Decostre V. Li J. Renou L. Kesari A. Hantai D. Stewart C.L. Xiao X. Hoffman E. Bonne G. Misteli T. Lamin A/C-mediated neuromuscular junction defects in Emery-Dreifuss muscular dystrophy.J Cell Biol. 2009; 184: 31-44Crossref PubMed Scopus (89) Google Scholar to deliver short-hairpin RNA (shRNA) to down-regulate FKRP expression in the skeletal muscles of wild-type ICR mice. We initially screened for small interference RNA (siRNA) oligonucleotides and shRNA capable of effectively knocking down FKRP expression in cultured cells. Subsequently, the selected shRNA sequences were cloned and packaged in AAV vectors and then delivered to the limb muscles of the animals by intramuscular injection. Our results showed that specific targeting of FKRP mRNA by RNAi reduced the level of FKRP transcripts in the skeletal muscle. Furthermore, long-term down-regulation of FKRP expression also resulted in hypoglycosylation of α-dystroglycan. AAV-shFKRP-vector–treated mice developed signs of chronic pathological changes associated with muscular dystrophy in limb muscles. Thus, postnatal knockdown of FKRP gene expression by means of AAV-mediated long-term transfer of shRNA is a useful approach for generating an LGMD2I mouse model and investigating the function of FKRP. Both Hepa 1–6 cells (a C57L mouse hepatoma cell line) and the 293 cells (a human embryonic kidney cell line) were grown in Dulbecco's modified Eagle's medium (Invitrogen, San Diego, CA) with antibiotics (penicillin and streptomycin; Invitrogen) and 10% heat-inactivated fetal bovine serum (HyClone, Thermo Scientific, Waltham, MA). For 293 cells, before the transfection, the density of cells was allowed to reach 80% to 85% in six-well culture plates. In each well, the transfection was followed by Lipofectamine with plus reagent method (Invitrogen). A total of 2 μg of plasmid DNA were applied to each well. After a 48-hour incubation, the protein was extracted and kept at -20°C for future testing. For Hepa 1–6 cells, the transfection was preceded by the DharmaFECT+ 1 method (Thermo Scientific). Briefly, the Hepa 1–6 cells were cultured to 80% confluence and trypsinized with trypsin-EDTA (Invitrogen) and counted; 5 × 105 cells were remixed with the mixture of siRNA (Integrated DNA Technologies) and the DharmaFECT 1 reagent. The final siRNA concentration per well was 100 nmol/L for 2.5 × 105 cells (24-well plate). It was incubated in a 37°C/5% CO2 incubator for 48 hours. After the first 24 hours of incubation, the medium was aspirated from the cells and replaced with 500 μL of fresh growth medium. The total RNA was extracted and kept at -80°C for further assay. All samples were prepared at least in triplicate, and the results were confirmed by independent experiments. All of the FKRP inhibitory 19-mer siRNA (total, 10 candidates) were designed as shRNA forms and cloned into an AAV vector driven by mouse RNA polymerase III promoters-U6 promoter, which was adapted from the commercial plasmid-pSilencer 1.0. The sequence TTCAAGAGA was used as the loop of shFKRP.49Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3963) Google Scholar For the FKRP2 shRNA construct, the sense (5′-GATCCCCGCACTTCTGTCCCGCTTCATTCAAGAGATGAAGCGGGACAGAAGTGCTTTTTGGA-3′) and antisense (5′-AGCTTCCAAAAAGCACTTCTGTCCCGCTTCATCTCTTGAATGAAGCGGGACAGAAGTGCGGG-3′) strands of shFKRP2 were designed with BamH I and HindIII restriction site linkers flanking the 5′ and 3′ termini of shRNA, respectively. Two oligos of 62 nucleotides, sense and antisense strands of the shRNA, were synthesized by Integrated DNA Technologies. The annealed shFKRP2 was cloned into the BamH I and HindIII sites on the double-stranded AAV vector plasmid containing the U6 promoter. Similarly for the FKRP5 shRNA construct, sense (5′-GATCCCCGCGACTTCTTCCGAGTACATTCAAGAGATGTACTCGGAAGAAGTCGCTTTTTGGA-3′) and antisense (5′-AGCTTCCAAAAAGCGACTTCTTCCGAGTACATCTCTTGAATGTACTCGGAAGAAGTCGCGGG-3′) strands of shFKRP5 were also cloned to the AAV vector. To test the additional effects of shFKRP2 and shFKRP5, a head-to tail dual-cassette vector (pAAV-U6-shFKRP2-U6-shFKRP5) was also constructed. A control vector pAAV-U6-shGFP has been described previously.48Mejat A. Decostre V. Li J. Renou L" @default.
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• W2040287583 date "2011-01-01" @default.
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• W2040287583 title "Post-Natal Knockdown of Fukutin-Related Protein Expression in Muscle by Long-Term RNA Interference Induces Dystrophic Pathology" @default.
• W2040287583 cites W1488929723 @default.
• W2040287583 cites W1519383882 @default.
• W2040287583 cites W1593294300 @default.
• W2040287583 cites W1968666078 @default.
• W2040287583 cites W1968990159 @default.
• W2040287583 cites W1969570221 @default.
• W2040287583 cites W1974360702 @default.
• W2040287583 cites W1975205104 @default.
• W2040287583 cites W1977222962 @default.
• W2040287583 cites W1978255162 @default.
• W2040287583 cites W1982268478 @default.
• W2040287583 cites W1985467308 @default.
• W2040287583 cites W1986132700 @default.
• W2040287583 cites W1989986119 @default.
• W2040287583 cites W1990956920 @default.
• W2040287583 cites W1997946559 @default.
• W2040287583 cites W2008115339 @default.
• W2040287583 cites W2008980136 @default.
• W2040287583 cites W2009991379 @default.
• W2040287583 cites W2014010760 @default.
• W2040287583 cites W2020785552 @default.
• W2040287583 cites W2021202438 @default.
• W2040287583 cites W2021233258 @default.
• W2040287583 cites W2023669917 @default.
• W2040287583 cites W2025349757 @default.
• W2040287583 cites W2029066095 @default.
• W2040287583 cites W2038783205 @default.
• W2040287583 cites W2041119826 @default.
• W2040287583 cites W2045156127 @default.
• W2040287583 cites W2046205597 @default.
• W2040287583 cites W2055378266 @default.
• W2040287583 cites W2055883057 @default.
• W2040287583 cites W2058955056 @default.
• W2040287583 cites W2061697489 @default.
• W2040287583 cites W2062006811 @default.
• W2040287583 cites W2065704959 @default.
• W2040287583 cites W2075506671 @default.
• W2040287583 cites W2077773751 @default.
• W2040287583 cites W2081535578 @default.
• W2040287583 cites W2083858363 @default.
• W2040287583 cites W2090667030 @default.
• W2040287583 cites W2092413879 @default.
• W2040287583 cites W2095998265 @default.
• W2040287583 cites W2099234787 @default.
• W2040287583 cites W2099278684 @default.
• W2040287583 cites W2099705607 @default.
• W2040287583 cites W2104254005 @default.
• W2040287583 cites W2106490838 @default.
• W2040287583 cites W2109073906 @default.
• W2040287583 cites W2111591442 @default.
• W2040287583 cites W2114100442 @default.
• W2040287583 cites W2120395097 @default.
• W2040287583 cites W2121650438 @default.
• W2040287583 cites W2123958892 @default.
• W2040287583 cites W2125293681 @default.
• W2040287583 cites W2125487882 @default.
• W2040287583 cites W2135383773 @default.
• W2040287583 cites W2136120716 @default.
• W2040287583 cites W2137162480 @default.
• W2040287583 cites W2139865704 @default.
• W2040287583 cites W2146711848 @default.
• W2040287583 cites W2146756838 @default.
• W2040287583 cites W2148223197 @default.
• W2040287583 cites W2149168171 @default.
• W2040287583 cites W2153776008 @default.
• W2040287583 cites W2160775842 @default.
• W2040287583 cites W2168506210 @default.
• W2040287583 cites W2168858900 @default.
• W2040287583 cites W2170867495 @default.
• W2040287583 cites W2171805399 @default.
• W2040287583 cites W2401520128 @default.
• W2040287583 cites W4249596337 @default.
• W2040287583 cites W4376453120 @default.
• W2040287583 doi "https://doi.org/10.1016/j.ajpath.2010.11.020" @default.
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