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Methods in Molecular Biology 2587 Rika Maruyama Toshifumi Yokota Editors Muscular Dystrophy Therapeutics Methods and Protocols M M B ETHODS IN OLECULAR IO LO GY SeriesEditor JohnM.Walker School of Lifeand MedicalSciences University ofHertfordshire Hatfield, Hertfordshire, UK Forfurther volumes: http://www.springer.com/series/7651 For over 35 years, biological scientists have come to rely on the research protocols and methodologiesinthecriticallyacclaimedMethodsinMolecularBiologyseries.Theserieswas thefirsttointroducethestep-by-stepprotocolsapproachthathasbecomethestandardinall biomedicalprotocolpublishing.Eachprotocolisprovidedinreadily-reproduciblestep-by- step fashion, opening with an introductory overview, a list of the materials and reagents neededtocompletetheexperiment,andfollowedbyadetailedprocedurethatissupported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitutethekeyingredientineachandeveryvolumeoftheMethodsinMolecularBiology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexedinPubMed. Muscular Dystrophy Therapeutics Methods and Protocols Edited by Rika Maruyama and Toshifumi Yokota Department of Medical Genetics, University of Alberta, Edmonton, AB, Canada Editors RikaMaruyama ToshifumiYokota DepartmentofMedicalGenetics DepartmentofMedicalGenetics UniversityofAlberta UniversityofAlberta Edmonton,AB,Canada Edmonton,AB,Canada ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2771-6 ISBN978-1-0716-2772-3 (eBook) https://doi.org/10.1007/978-1-0716-2772-3 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2023 Thisworkissubjecttocopyright.AllrightsaresolelyandexclusivelylicensedbythePublisher,whetherthewholeorpart of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting,reproductionon microfilmsorinanyotherphysicalway,andtransmissionorinformation storageand retrieval,electronicadaptation, computersoftware,orbysimilar ordissimilar methodologynow knownorhereafter developed. Theuseofgeneraldescriptivenames,registerednames,trademarks,servicemarks,etc.inthispublicationdoesnotimply, evenintheabsenceofaspecificstatement,thatsuchnamesareexemptfromtherelevantprotectivelawsandregulations andthereforefreeforgeneraluse. Thepublisher,theauthors,andtheeditorsaresafetoassumethattheadviceandinformationinthisbookarebelievedto betrueandaccurateatthedateofpublication.Neitherthepublishernortheauthorsortheeditorsgiveawarranty, expressedorimplied,withrespecttothematerialcontainedhereinorforanyerrorsoromissionsthatmayhavebeen made.Thepublisherremainsneutralwithregardtojurisdictionalclaimsinpublishedmapsandinstitutionalaffiliations. ThisHumanaimprintispublishedbytheregisteredcompanySpringerScience+BusinessMedia,LLC,partofSpringer Nature. Theregisteredcompanyaddressis:1NewYorkPlaza,NewYork,NY10004,U.S.A. Preface “The greatest enemy of knowledge is not ignorance, it is the illusion of knowledge”-StephenHawking With the development of novel targeted therapies, including antisense-mediated exon skipping and gene replacement therapy, the field of muscular dystrophy therapeutics has been drastically changed in the last several years. Until 2016, there had been no FDA-approved drug to treat Duchenne muscular dystrophy (DMD), the most common muscular dystrophy. However, several new therapies, including antisense oligonucleotides, small molecules, and gene replacement therapy, are now approved or under late-stage clinicaltrials.Wearewitnessingthestartofatherapeuticrevolutioninmusculardystrophy. However,thestudiesalsomadeclear thatthesetherapiesarestillfar fromacure. Thisbookpresentsacomprehensivecollectionofstate-of-the-artprotocolsonmuscular dystrophy therapeutics, covering therapeutics using antisense oligonucleotides, gene replacement,genomeediting,smallmolecules,stemcells,andantibodies,fromtheleaders inthefield.Thegoalofthisbookistoinspirereadersandprovidehands-ontips,strategies, andadvicetodevelopnewtherapeutictechnologiesfor musculardystrophy. We are very grateful to all authors for sharing their detailed methods. These detailed methods and tips arenot easily available elsewhere.Wealso deeply thank Dr. John Walker, the series editor, for his excellent guidance, careful paper reading, and encouragement throughout the development of this book. We are very grateful to the Friends of Garrett CummingResearch,MuscularDystrophyCanada,HenriM.ToupinChairinNeurological Sciences, and the University of Alberta for the funding to establish a muscular dystrophy researchlabattheUniversityofAlberta.WearealsothankfultotheCanadianInstitutesof HealthResearch (CIHR),Canada Foundationfor Innovation(CFI), Japan Societyfor the Promotion of Science (JSPS), Alberta Enterprise and Advanced Education, BC Children’s Hospital Foundation, FSH Society, FSHD Canada Foundation, Rare Disease Foundation, and Defeat Duchenne Foundation (Jesse’s Journey) for their support in expanding our research in developing antisense therapies for muscular dystrophy. Lastly but most impor- tantly,wearedeeplygratefultothepatientsandfamilymembers;theirsupport,inspiration, andparticipationinresearchhavebeencriticaltothedevelopmentofnoveltherapies. Edmonton,AB,Canada RikaMaruyama ToshifumiYokota v Contents Preface ..................................................................... v Contributors................................................................. xi PART I BASICS AND INTRODUCTION 1 CurrentStrategiesofMuscularDystrophyTherapeutics:AnOverview ........ 3 KenjiRowelQ.LimandToshifumiYokota 2 Viltolarsen:FromPreclinicalStudiestoFDAApproval ....... ....... ........ 31 RohiniRoyRoshmiandToshifumiYokota PART II ANIMAL MODELS, SAMPLE PREPARATION, AND ASSESSMENT 3 RapidFreezingofSkeletalandCardiacMusclesUsingIsopentaneCooled withLiquidNitrogenandTragacanthGumforHistological,Genetic, andProteinExpressionStudies ...... ....... ....... ........ ....... ........ 45 SaeedAnwarandToshifumiYokota 4 CardiacandSkeletalMusclePathologyintheD2/mdxMouseModel andCaveatsAssociatedwiththeQuantificationofUtrophin ......... ........ 55 TahneeL.KennedyandHannahF.Dugdale 5 PhysiologicalAssessmentofMuscle,Heart,andWholeBodyFunction intheCanineModelofDuchenneMuscularDystrophy ...... ....... ....... . 67 ChadyH.Hakim,JamesTeixeira,StacyB.Leach,andDongshengDuan PART III ANTISENSE OLIGONUCLEOTIDES AND GENE REPLACEMENT THERAPIES 6 RestoringDystrophinExpressionbySkippingExons6and8 inNeonatalDystrophicDogs........ ....... ....... ........ ....... ........ 107 MdNurAhadShahandToshifumiYokota 7 RestoringDystrophinExpressionwithExon44and53Skipping intheDMDGeneinImmortalizedMyotubes....... ........ ....... ........ 125 YusukeEchigoyaandToshifumiYokota 8 RestoringDystrophinExpressionwithDuchenneMuscularDystrophy Exon45SkippinginInducedPluripotentStemCell-Derived Cardiomyocytes..... ....... ........ ....... ..... .. ... ..... ....... ........ 141 MitsutoSato,NaokoShiba,DaigoMiyazaki, YujiShiba,andAkinoriNakamura 9 QuantitativeEvaluationofExonSkippinginUrine-Derived CellsforDuchenneMuscularDystrophy..... ....... ........ ....... ........ 153 KatsuhikoKunitake,ChaitraSathyaprakash, NorioMotohashi,andYoshitsuguAoki vii viii Contents 10 UseofGlycinetoAugmentExonSkippingandCellTherapies forDuchenneMuscularDystrophy ......... ....... ........ ....... ........ 165 GangHan,CaoruiLin,andHaiFangYin 11 Morpholino-MediatedExons28–29SkippingofDysferlin andCharacterizationofMultiexon-skippedDysferlin usingRT-PCR,Immunoblotting,andMembraneWoundingAssay ........... 183 SaeedAnwarandToshifumiYokota 12 KnockingDownDUX4inImmortalizedFacioscapulohumeral MuscularDystrophyPatient-DerivedMuscleCells........... ....... ........ 197 KenjiRowelQ.LimandToshifumiYokota 13 Peptide-ConjugatedPMOsfor theTreatmentofMyotonicDystrophy........ 209 JessicaStoodley,DavidSeoaneMiraz,YahyaJad, MathieuFischer,MatthewJ.A.Wood,andMiguelA.Varela 14 DevelopingTherapeuticSplice-CorrectingAntisenseOligomers forAdult-OnsetPompeDiseasewithc.-32-13T>GMutation........ ........ 239 KristinA.Ham,RussellD.Johnsen,MichelTchan, SteveD.Wilton,andMayT.Aung-Htut PART IV GENE REPLACEMENT THERAPIES 15 MolecularandBiochemicalAssessmentofGeneTherapy intheCanineModelofDuchenneMuscularDystrophy...... ....... ........ 255 ChadyH.Hakim,DennisPe´rez-L(cid:1)opez,MatthewJ.Burke, JamesTeixeira,andDongshengDuan 16 HistologicalAssessmentofGeneTherapyintheCanineDMDModel ........ 303 ChadyH.Hakim,MatthhewJ.Burke,JamesTeixeira, andDongshengDuan 17 MRIEvaluationofGeneTherapyintheCanineModel ofDuchenneMuscularDystrophy .......... ....... ........ ....... ........ 339 AmyR.Zalcman,ChadyH.Hakim,JimmyC.Lattimer, JamesR.Holland,JohnR.Dodam,andDongshengDuan 18 AssessmentoftheGeneTherapyImmuneResponseintheCanine MuscularDystrophyModel......... ....... ....... ........ ....... ....... . 353 ChadyH.Hakim,SandeepR.P.Kumar,DennisPe´rez-L(cid:1)opez, JamesTeixeira,RolandW.Herzog,andDongshengDuan 19 UseofMesenchymalStemCellstoEnhancetheEfficacy ofGeneTherapy.... ....... ........ ....... ....... ........ ....... ........ 377 HiromiHayashita-KinohandTakashiOkada 20 Exon-SkippingforaPathogenicCOL6A1VariantinUllrich CongenitalMuscularDystrophy..... ....... ....... ........ ....... ........ 387 SaraAguti,FadyGuirguis,CarstenBönnemann, FrancescoMuntoni,Ve´roniqueBolduc,andHaiyanZhou Contents ix PART V GENOME EDITING/CRISPR 21 CRISPR-Cas9CorrectionofDuchenneMuscularDystrophyinMice byaSelf-ComplementaryAAVDeliverySystem ..... ........ ....... ........ 411 YuZhang,RhondaBassel-Duby,andEricN.Olson 22 PreparationofNanoMEDICExtracellularVesiclestoDeliver CRISPR-Cas9RibonucleoproteinsforGenomicExonSkipping...... ........ 427 KeiWatanabe,PeterGee,andAkitsuHotta 23 RestorationofDystrophinExpressioninMdx-DerivedMuscleProgenitor CellsUsingCRISPR/Cas9SystemandHomology-Directed RepairTechnology......... ........ ....... ....... ........ ....... ........ 455 YueJin,YanShen,Il-manKim,NealL.Weintraub, MarkHamrick,andYaoliangTang PART VI SMALL MOLECULES 24 EffectsofGlucocorticoidsinMurineModelsofDuchenne andLimb-GirdleMuscularDystrophy....... ...... ....... .. ...... .... ..... 467 MichelleWintzinger,KarenMiz,AllenYork,AlexisR.Demonbreun, JefferyD.Molkentin,ElizabethM.McNally,andMattiaQuattrocelli 25 High-ThroughputScreeningtoIdentifyModulatorsofSarcospan.... ........ 479 CynthiaShu,EkaterinaMokhonova,andRachelleH.Crosbie 26 IdentifyingFDA-ApprovedDrugsthatUpregulateUtrophinA asaTherapeuticStrategyforDuchenneMuscularDystrophy......... ........ 495 ChristinePe´ladeauandBernardJ.Jasmin PART VII CELL MODELS AND STEM CELLS 27 MonitoringPlasmaMembraneInjury-TriggeredEndocytosis atSingle-CellandSingle-VesicleResolution......... ........ ....... ........ 513 DanielC.BittelandJyotiK.Jaiswal 28 EvaluationofhiPSC-DerivedMuscleProgenitorCellTransplantation inaMouseDuchenneMuscularDystrophyModel........... ....... ....... . 527 MinasNalbandian,MingmingZhao,andHidetoshiSakurai 29 QuantificationofMuscleSatelliteStemCellDivisions byHigh-ContentAnalysis .......... ....... ....... ........ ....... ........ 537 WilliamChen,TheodoreJ.Perkins,andMichaelA.Rudnicki PART VIII ANTIBODY 30 SystemicDeliveryofaMonoclonalAntibodytoImmunologically BlockMyostatinintheA17MouseModelofOPMD........ ....... ........ 557 AlbertoMalerba,PradeepHarish,andLindaPopplewell Index ...................................................................... 569 Contributors SARAAGUTI • TheDubowitzNeuromuscularCentre,MolecularNeurosciencesSection, DevelopmentalNeurosciencesResearchandTeachingDepartment,GreatOrmondStreet InstituteofChildHealth,UniversityCollegeLondon,London,UK SAEEDANWAR • DepartmentofMedicalGenetics,FacultyofMedicineandDentistry, UniversityofAlberta,Edmonton,AB,Canada YOSHITSUGU AOKI • DepartmentofMolecularTherapy,NationalInstituteofNeuroscience, NationalCenterofNeurologyandPsychiatry(NCNP),Tokyo,Japan MAYT.AUNG-HTUT • CentreforMolecularMedicineandInnovativeTherapeutics,Health FuturesInstitute,MurdochUniversity,Murdoch,WA,Australia;PerronInstitutefor NeurologicalandTranslationalScience,Nedlands,WA,Australia;Centrefor NeuromuscularandNeurologicalDisorders,UniversityofWesternAustralia,Crawley, WA,Australia RHONDABASSEL-DUBY • DepartmentofMolecularBiology,UniversityofTexasSouthwestern MedicalCenter,Dallas,TX,USA;HamonCenterforRegenerativeScienceandMedicine, UniversityofTexasSouthwesternMedicalCenter,Dallas,TX,USA;SenatorPaul D.WellstoneMuscularDystrophySpecializedResearchCenter,UniversityofTexas SouthwesternMedicalCenter,Dallas,TX,USA DANIELC.BITTEL • Center forGeneticMedicineResearch,Children’sNationalResearch Institute,Washington,DC,USA VE´RONIQUEBOLDUC • NeuromuscularandNeurogeneticDisordersofChildhoodSection, NationalInstituteofNeurologicalDisordersandStroke,NationalInstitutesofHealth, Bethesda,MD,USA CARSTENBO¨NNEMANN • NeuromuscularandNeurogeneticDisordersofChildhoodSection, NationalInstituteofNeurologicalDisordersandStroke,NationalInstitutesofHealth, Bethesda,MD,USA MATTHHEWJ.BURKE • DepartmentofMolecularMicrobiologyandImmunology,Schoolof Medicine,TheUniversityofMissouri,Columbia,MO,USA WILLIAMCHEN • SprottCenter forStemCellResearch,RegenerativeMedicineProgram, OttawaHospitalResearchInstitute,Ottawa,ON,Canada;DepartmentofCellularand MolecularMedicine,FacultyofMedicine,UniversityofOttawa,Ottawa,ON,Canada RACHELLEH.CROSBIE • DepartmentofIntegrativeBiologyandPhysiology,Universityof CaliforniaLosAngeles,LosAngeles,CA,USA;DepartmentofNeurology,DavidGeffen SchoolofMedicine,UniversityofCaliforniaLosAngeles,LosAngeles,CA,USA ALEXISR.DEMONBREUN • Center forGeneticMedicine,FeinbergSchoolofMedicine, NorthwesternUniversity,Chicago,IL,USA JOHNR.DODAM • DepartmentofVeterinaryMedicineandSurgery,CollegeofVeterinary Medicine,TheUniversityofMissouri,Columbia,MO,USA DONGSHENGDUAN • DepartmentofMolecularMicrobiologyandImmunology,Schoolof Medicine,TheUniversityofMissouri,Columbia,MO,USA;DepartmentofBiomedical, Biological&ChemicalEngineering,CollegeofEngineering,TheUniversityofMissouri, Columbia,MO,USA;DepartmentofNeurology,SchoolofMedicine,TheUniversityof Missouri,Columbia,MO,USA;DepartmentofBiomedicalSciences,CollegeofVeterinary Medicine,TheUniversityofMissouri,Columbia,MO,USA xi