Methods in Molecular Biology 2573 Kiyotake Ishikawa Editor Cardiac Gene Therapy Methods and Protocols Second Edition 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 biomedical protocol publishing. Each protocol is provided in readily-reproducible step-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. 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Cardiac Gene Therapy Methods and Protocols Second Edition Edited by Kiyotake Ishikawa Icahn School of Medicine at Mount Sinai, New York, NY, USA Editor KiyotakeIshikawa IcahnSchoolofMedicineatMount Sinai NewYork,NY,USA ISSN1064-3745 ISSN1940-6029 (electronic) MethodsinMolecularBiology ISBN978-1-0716-2706-8 ISBN978-1-0716-2707-5 (eBook) https://doi.org/10.1007/978-1-0716-2707-5 ©TheEditor(s)(ifapplicable)andTheAuthor(s),underexclusivelicensetoSpringerScience+BusinessMedia,LLC,part ofSpringerNature2022 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. 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Preface SincethepublicationofCardiacGeneTherapyin2016,therehasbeenconsiderableprogress in clinical translation of gene therapy, highlighted by clinical approvals for multiple gene therapyproducts.Unfortunately,nogenetherapyspecificallytargetingthehearthastrans- latedintoclinicalpracticeyet,andtodate,onlyafewearly-phaseclinicaltrialsareunderway. However, activity in preclinical areas of cardiac gene therapy has gained momentum and is rapidly growing toward the realization of next-generation therapy for cardiovascular diseases. In the second edition of Cardiac Gene Therapy, I recruited authors with long-term expertise in cardiac gene therapy experiments. Many of the collected protocol chapters are from expert investigators who have published novel work in the past few years since the publication of the first edition. With a focus on promoting further clinical translation, coveredtopicsincludegenesuppression,editing,andreprogramming;cardiacgenetherapy vectors and promotors; cardiac gene delivery methods; pulmonary hypertension; and patient screening and measuring the efficacy of cardiac gene therapy. Together with the protocolsincludedinthefirstedition,thesedetailedandpracticalprotocolswillbevaluable toolsfor researchersincardiologytoconductcardiacgenetherapyresearch. Ithankalltheexpertauthorsfor theirdedicationindescribingstep-by-stepmethodol- ogiesthatwillundoubtedlyleadtofurtheradvancementsandsuccessfulclinicaltranslation of cardiac gene therapy. I am incredibly grateful to John M. Walker, the series editor, who providedmewiththisopportunityandguidedthevolumepreparationprocess.Ihopethat readers will find Cardiac Gene Therapy: Methods and Protocols, Second Edition a useful referenceforconductingandimprovingtheirprojects. NewYork,NY,USA KiyotakeIshikawa v Contents Preface ..................................................................... v Contributors................................................................. xi PART I OVERVIEW 1 UpdatesonCardiacGeneTherapyResearchandMethods:Overview ofCardiacGeneTherapy ........... ....... ....... ........ ....... ........ 3 FranciscoJ.Romeo,SpyrosA.Marvopoulos, andKiyotakeIshikawa PART II GENE SUPPRESSION, EDITING, AND REPROGRAMMING 2 ToughDecoy-MediatedCardiacGeneSuppression .......... ....... ........ 13 ChangwonKho 3 DirectReprogrammingofAdultHumanCardiacFibroblasts intoInducedCardiomyocytesUsingmiRcombo..... ........ ....... ........ 31 CamillaPaoletti,CarlaDivieto,andValeriaChiono 4 CRISPR/Cas9GeneEditingofRYR2inHumaniPSC-Derived CardiomyocytestoProbeCa2+SignalingAberrancies ofCPVTArrhythmogenesis........ ....... ....... ... ...... ....... ........ 41 NaohiroYamaguchi,Xiao-HuaZhang,andMartinMorad 5 EnhancingCardiomyocyteTranscriptionUsingInVivo CRISPR/Cas9Systems..... ........ ....... ....... ........ ....... ........ 53 EricSchogerandLauraC.Zelaraya´n 6 AAV-MediatedSomaticGeneEditingforCardiacandSkeletal MuscleinaLargeAnimalModel..... ....... ....... ........ ....... ...... .. 63 TilmanZiegler,TarikBozoglu,andChristianKupatt PART III CARDIAC GENE THERAPY VECTORS AND PROMOTORS 7 OptimizationofSynthesisofModifiedmRNA....... ........ ....... ........ 77 JimeenYooandLiorZangi 8 DesignandProductionofHeartChamber-SpecificAAV9Vectors .... ........ 89 AlinaS.Bilal,DonnaJ.Thuerauf,ErikA.Blackwood, andChristopherC.Glembotski 9 GenerationofAtrial-SpecificConstructUsingSarcolipin Promoter-AssociatedCRM4Enhancer ...... ....... ........ ....... ........ 115 DongtakJeong vii viii Contents PART IV CARDIAC GENE DELIVERY METHODS 10 CardiacTargetedAdeno-AssociatedVirusInjectioninRats.......... ........ 135 MichaelG.Katz,YoavHadas,AdamS.Vincek, NatalyShtraizent,EricSchadt,andEfratEliyahu 11 CardiacGeneDeliveryinLargeAnimalModels: AntegradeTechniques...... ........ ....... ....... ........ ....... ........ 147 SpyrosA.Mavropoulos,KellyP.Yamada,TomokiSakata, andKiyotakeIshikawa 12 LockedNucleicAcidAntimiRTherapyfor theHeart ........ ....... ........ 159 SabineSamolovacandRabeaHinkel 13 CardiacGeneDeliveryinLargeAnimalModels:SelectiveRetrograde VenousInjection.... ....... ........ .. ..... ...... ....... .. ....... ........ 171 PhilippSchlegelandPhilipW.J.Raake 14 EndocardialGeneDeliveryUsingNOGACatheterSystem .......... ........ 179 SatuSiimes,NikoJa€rvela€inen,HennaKorpela, andSeppoYla€-Herttuala 15 SurgicalMethodsforCardiacGeneDeliveryinLargeAnimals ....... ........ 189 MichaelG.Katz,YoavHadas,AdamS.Vincek,NatalyShtraizent, HyltonP.Gordon,PeterPastuszko,EricSchadt,andEfratEliyahu 16 AtrialGenePaintinginLargeAnimalModelofAtrialFibrillation..... ........ 205 WeilanMoandJ.KevinDonahue 17 Stent-BasedGeneDeliveryforCoronaryDisease .... ........ ....... ........ 217 IvanS.Alferiev,MichaelChorny,RobertL.Wilensky, RobertJ.Levy,andIliaFishbein 18 SelectiveAnti-AAVAntibodyDepletionbyHemapheresis andImmunoadsorption .... ...... .. ....... ....... ........ ....... ........ 235 AlejandroOrlowskiandThomasWeber 19 ExVivoDeliveryofViralVectorsbyOrganPerfusion forCardiacTransplantationGeneTherapy.......... ........ ....... ........ 249 MichelleMendiolaPla,AmyEvans,PaulLezberg, andDawnE.Bowles PART V PULMONARY HYPERTENSION 20 Intra-AirwayGeneDeliveryforPulmonaryHypertension inRodentModels.......... ........ ....... ....... ....... .... .... ........ 263 MalikBisserier,OlivierBoucherat,SebastienBonnet, andLahouariaHadri 21 EndobronchialGeneDeliveryforPulmonaryHypertension inaLargeAnimalModel ........... ....... ....... ........ ....... ........ 279 OlympiaBikouandKiyotakeIshikawa Contents ix PART VI PATIENT SCREENING AND MEASURING THE EFFICACY OF CARDIAC GENE THERAPY 22 Cell-BasedDeterminationofNeutralizingAntibodiesAgainst Adeno-AssociatedVirusinCardiacGeneTherapy ........... ....... ........ 293 AnjaliJ.Ravichandran,RenataMazurek, andKiyotakeIshikawa 23 LeftVentricularPressureVolumeAssessmentUsingCarotid ArteryAccessintheRat .... ........ ....... ....... ........ ....... ........ 305 SpyrosA.MavropoulosandKiyotakeIshikawa 24 AssessingtheEffectofCardiacGeneTherapyUsingCatheter-Based Pressure–VolumeMeasurementinLargeAnimals.... ........ ....... ........ 313 TomokiSakata,RenataMazurek,SpyrosA.Mavropoulos, FranciscoJ.Romeo,AnjaliJ.Ravichandran, andKiyotakeIshikawa 25 ELISpotAssayforGeneTherapyinLargeAnimalStudies .... ...... ..... .... 323 RenataMazurekandKiyotakeIshikawa 26 AssessingRecombinantAAVSheddingAfterCardiacGeneTherapy .......... 333 MeladFarrahaandEddyKizana Index ...................................................................... 345 Contributors IVANS.ALFERIEV • TheChildren’sHospitalofPhiladelphia,Philadelphia,PA,USA;The UniversityofPennsylvaniaPerelmanSchoolofMedicine,Philadelphia,PA,USA OLYMPIABIKOU • CardiovascularResearchInstitute,IcahnSchoolofMedicineatMount Sinai,NewYork,NY,USA ALINAS.BILAL • TranslationalCardiovascularResearchCenterandDepartmentof InternalMedicine,UniversityofArizonaCollegeofMedicine–Phoenix,Phoenix,AZ, USA MALIKBISSERIER • CardiovascularResearchInstitute,IcahnSchoolofMedicineatMount Sina,NewYork,NY,USA ERIK A.BLACKWOOD • TranslationalCardiovascularResearchCenterandDepartmentof InternalMedicine,UniversityofArizonaCollegeofMedicine–Phoenix,Phoenix,AZ, USA SEBASTIENBONNET • PulmonaryHypertensionResearchGroup,Que´becHeartandLung InstituteResearchCentre,QC,Canada;DepartmentofMedicine,LavalUniversity,QC, Canada OLIVIER BOUCHERAT • PulmonaryHypertensionResearchGroup,Que´becHeartandLung InstituteResearchCentre,QC,Canada;DepartmentofMedicine,LavalUniversity,QC, Canada DAWNE.BOWLES • DukeUniversity,Durham,NC,USA TARIKBOZOGLU • KlinikundPoliklinikfu¨rInnereMedizinI,KlinikumrechtsderIsarder TechnicalUniversityMunich,Munich,Germany;GermanCenter forCardiovascular Research(DZHK),MunichHeartAlliance,Munich,Germany VALERIA CHIONO • DepartmentofMechanicalandAerospaceEngineering,Politecnicodi Torino,Turin,Italy;DepartmentforMaterialsandDevicesoftheNationalResearch Council,Institutefor theChemicalandPhysicalProcesses(CNR-IPCFUOS),Pisa,Italy MICHAELCHORNY • TheChildren’sHospitalofPhiladelphia,Philadelphia,PA,USA;The UniversityofPennsylvaniaPerelmanSchoolofMedicine,Philadelphia,PA,USA CARLADIVIETO • DivisionofAdvancedMaterialsandLifeSciences,IstitutoNazionaledi RicercaMetrologica,Turin,Italy J.KEVINDONAHUE • CardiovascularMedicine,UMassChanMedicalSchool,Worcester,MA, USA EFRATELIYAHU • DepartmentofGeneticsandGenomicSciences,IcahnInstitutefor GenomicsandMultiscaleBiology,IcahnSchoolofMedicineatMountSinai,NewYork,NY, USA;IcahnSchoolforDataScienceandGenomicTechnology,IcahnSchoolofMedicineat MountSinai,NewYork,NY,USA AMYEVANS • DukeUniversity,Durham,NC,USA MELADFARRAHA • CentreforHeartResearch,TheWestmeadInstituteforMedicalResearch, Westmead,NSW,Australia ILIAFISHBEIN • TheChildren’sHospitalofPhiladelphia,Philadelphia,PA,USA;The UniversityofPennsylvaniaPerelmanSchoolofMedicine,Philadelphia,PA,USA CHRISTOPHERC.GLEMBOTSKI • TranslationalCardiovascularResearchCenterand DepartmentofInternalMedicine,UniversityofArizonaCollegeofMedicine–Phoenix, Phoenix,AZ,USA xi