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Lentiviral Integrase-Fusions: Genomic Interactions, Site Selection

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Lentiviral Integrase-Fusions: Genomic Interactions, Site Selection dissertations | 167 | Vesa| 167 Turkki | Vesa Turkki Lentiviral Integrase-fusions: Genomic Interactions, In this study, a protein delivery Site Selection and Cellular method based on lentiviral Lentiviral Integrase-fusions: Genomic Interactions, Site Selection and Cellular Delivery Heterologous of Proteins Vesa Turkki integrase-fusions was characterized. Delivery of Heterologous Its applicability was demonstrated Proteins with a fluorescent marker protein Lentiviral Integrase-fusions: Genomic to allow virus labeling, a tumour Interactions, Site Selection and Cellular suppressor protein and a DNA- cleaving meganuclease. Modified Delivery of Heterologous Proteins cleavage-deficient meganucleases were shown capable of directing the integration into genomic target areas. Furthermore, the interactions between the integrase and the host cell genome were mapped for the first time. Publications of the University of Eastern Finland Dissertations in Health Sciences Publications of the University of Eastern Finland Dissertations in Health Sciences isbn 978-952-61-1113-1 VESA TURKKI Lentiviralintegrasefusions:Genomic interactions,siteselectionandcellular deliveryofheterologousproteins = = = = TobepresentedbypermissionoftheFacultyofHealthSciences,UniversityofEasternFinlandfor publicexaminationinTietotekniaauditorioum,Kuopio,onSaturday,May25th==2013,at12noon = = PublicationsoftheUniversityofEasternFinland= DissertationsinHealthSciences Number167 = = DepartmentofBiotechnologyandMolecularMedicine,A.I.VirtanenInstituteforMolecular Sciences,FacultyofHealthSciences,UniversityofEasternFinland= Kuopio= 2013= = = = = = = = = = = = = = = = = = = = = = = KopijyväOy Kuopio,2013 = SeriesEditors: ProfessorVeliMattiKosma,M.D.,=Ph.D. InstituteofClinicalMedicine,Pathology FacultyofHealthSciences = ProfessorHanneleTurunen,Ph.D. DepartmentofNursingScience= FacultyofHealthSciences = ProfessorOlliGröhn,Ph.D. A.I.VirtanenInstituteforMolecularSciences= FacultyofHealthSciences = Distributor: UniversityofEasternFinland KuopioCampusLibrary P.O.Box1627 FI70211Kuopio,Finland http://www.uef.fi/kirjasto = ISBN(print):9789526111131= ISBN(pdf):9789526111148 ISSN(print):17985706 ISSN(pdf):17985714 ISSNL:17985706 III Author’saddress:= DepartmentofBiotechnologyandMolecularMedicine A.I.VirtanenInstituteforMolecularSciences= UniversityofEasternFinland P.O.Box1627 FI70211KUOPIO FINLAND= [email protected] = Supervisors: ProfessorSeppoYläHerttuala,M.D.,Ph.D.= DepartmentofBiotechnologyandMolecularMedicine A.I.VirtanenInstituteforMolecularSciences= UniversityofEasternFinland = MikkoTurunen,Ph.D. DepartmentofBiotechnologyandMolecularMedicine A.I.VirtanenInstituteforMolecularSciences= UniversityofEasternFinland = = Reviewers: VarpuMarjomäki,Ph.D DivisionofCellandMolecularBiology NanoscienceCenter= DepartmentofBiologicalandEnvironmentalScience UniversityofJyväskylä JYVÄSKYLÄ FINLAND= = MarkusVähäKoskela,Ph.D DepartmentofPathologyandTransplantationlaboratory HaartmanInstitute UniversityofHelsinki HELSINKI= FINLAND= = Opponent: ProfessorArtoUrtti,Ph.D. CentreforDrugResearch FacultyofPharmacy UniversityofHelsinki HELSINKI= FINLAND= == IV = V Turkki,Vesa Lentiviral integrasefusions: Genomic= interactions, site selection and cellular delivery of heterologous proteins,89p.= Universityof=EasternFinland,FacultyofHealthSciences,2013 PublicationsoftheUniversityofEasternFinland.DissertationsinHealthSciences167.2013.89p. = ISBN(print):9789526111131 ISBN(pdf):9789526111148 ISSN(print):=17985706 ISSN(pdf):17985714 ISSNL:17985706 = ABSTRACT Ingenetherapy,newgeneticmaterialwhoseexpressionimprovesthestatusofpatient,is transportedintotargetcells,usuallybymeansofagenecarriercalleda=vector.Overthe= years,aconsiderablenumberoftreatmentstrategiesandavarietyoftransgeneshavebeen proposedforuseingenetherapyapplications.Unfortunatelyseveralpromisingconcepts havefailedduetothelackofsafeandefficientgenetransfervector.Lentiviralvectorsarea feasible option, especially for therapies requiring longterm transgene expression. This is due to the permanent integration of the transgene into the host cell’s genome, a reaction catalyzed by the viral integrase protein.= Another lentiviral application 8= transport of= functionalproteinsinsteadoftheircodingDNAs8=isanefficienttoolforresearchaswellas analternativetherapeuticapproach. = In this work a novel cispackaging strategy for heterologous protein incorporation into thirdgeneration lentiviral vectors was characterized. The method relies on integrase fusion, which was shown here to be tolerated in these vectors despite earlier negative= indications.PackagingoffusionproteinswasdemonstratedusingfluorescentmCherryand tumour suppressor p53 as fusion partners.= In the present studies, vectors and viruslike particles exhibited the correct proteolytic processing, fusion activity and transgene= integration. = Thegenomicdistributionofintegrationsites=hasamajorimpactonthesafetyprofileof=the= vectors. For targeted integration studies, an integrase fusion protein with a sequence specificIPpoImeganucleasewasconstructedalongwithfunctionalmutantsfordifferent applications. The meganuclease, when stripped of its cutting capability,= but without interfering with DNArecognition, was shown to be able to enhance integration into its= naturaltargetareasinthe=genome.InordertofurthercharacterizetheeffectsofIPpoI– induced double strand breaks, several cell lines were studied. Variable responses were= observedintermsofcytotoxicity.Inparticular,cancercellsweresusceptibletotreatment andthisapparentcytotoxiceffectwaspreservedinaninvivo=tumourstudy.Thespecificity ofIPpoIcleavagewasdemonstratedbychromatinimmunoprecipitationbasedstrategies, where the sites of interactions= between viral integrase and the host chromatin were mapped.Alsointerestingresultswarrantingfuturestudieswereobtainedintermsofthe= interaction and integration patterns of wildtype IN. In summary the results obtained in thisthesishighlightthebenefitsofthedirectcispackagingmethodandprovideproofof concept evidence for integrasefusion protein mediated transgene targeting and protein transduction. = NationalLibraryofMedicineClassification:QU470,QZ52,QW=51,QW168.5.R18,QW160 MedicalSubjectHeadings:=GeneTransferTechniques;GeneTherapy;Transduction,Genetic;GeneticVectors; Lentivirus;HIVIntegrase;ViralFusionProteins;GeneTargeting= VI = VII Turkki,Vesa Lentiviruksen integraasifuusiot: Genomiset interaktiot,= integraation kohdealueet ja vieraiden proteiinien= solukuljetus,89s.= ItäSuomenyliopisto,terveystieteidentiedekunta,2013. ItäSuomenyliopistonjulkaisuja.Terveystieteidentiedekunnanväitöskirjat167.2013.89s. = ISBN(print):9789526111131 ISBN(pdf):9789526111148 ISSN(print):=17985706 ISSN(pdf):17985714 ISSNL:17985706 = TIIVISTELMÄ Geeniterapiassa potilaaseen siirretään uutta geneettistä materiaalia, jonka ilmentäminen kudoksissajohtaaterapeuttiseenvaikutukseen.Geneettisenmateriaalinsiirtoonkäytetään vektoreiksi kutsuttuja geenikuljettimia, jotka on usein muokattu virusten pohjalta. Geeniterapiakokeita varten on tutkittu useita erilaisia siirtogeenejä, mutta monet lupaavatkaanlähestymistavateivätoletuottaneettoivottuavaikutustataiovatjohtaneetei toivottuun lopputulokseen optimaalisen vektorin puutteessa. Lentiviruksista voidaan kehittää vektoreita= erityisesti pitkäaikaista siirtogeenin ilmentymistä vaativiin hoitomuotoihin. Lentivirusvektoreilla on kyky liittää,= eli integroida, siirtogeeni kiinteäksi osaksi kohdesolun genomia, saaden aikaan siten jopa pysyvän geenin ilmentymisen.= Geeninliittämisestägenomiinvastaavektorinintegraasiproteiini.=Toinenlentivirussovellus= niin tutkimus kuin hoitokäyttöön on siirtää kohdesoluihin geenin sijasta suoraan sen= ilmentämääproteiinia(nk.proteiinitransduktio). = Työssä esiteltiin uusi suora pakkausmenetelmä vieraiden proteiinien liittämiseksi kolmannen sukupolven lentivirusvektoreihin. Työssä osoitettiin, että aiemmista negatiivista indikaatioista huolimatta kuljetettavat proteiinit voidaan fuusioida vektorin integraasiin. Pakkausmenetelmän toimivuus osoitettiin sekä fluoresoivan mCherry että tuumorien kasvua inhiboivan p53proteiinin avulla.= Tuotetut vektorit sisälsivät oikein prosessoituja, aktiivisia proteiineja ja niiden integraasit kykenivät integroimaan siirtogeenejäkohdesolungenomiin.Siirtogeenienintegraatiopaikkojenjakaumallaonsuuri merkitys vektorin potilasturvallisuuteen. Työssä osoitettiin myös mahdolliseksi muuttaa= lentivirusvektorin integraatiopaikkajakaumaa liittämällä integraasiin spesifisiä DNA8 alueita tunnistava ja leikkaava IPpoImeganukleaasi. Integraation kohdentamista varten meganukleaasiamuokattiinpoistaensenkykyleikata=DNA:ta.Muutoksenjälkeenvektori kykeni yhä ohjaamaan integraatiota kohdealueilleen eli ribosomin RNArakennetta= koodaaviin genomin osiin. Leikkaavaa IPpoI:tä sisältävää vektoria tutkittiin useissa solulinjoissa sen solutoksisten vaikutusten selvittämiseksi. Suurilla annoksilla vektori oli solutoksinenkaikissasoluissaeräidensyöpäsolulinjojenreagoidessaerityisenvoimakkaasti= käsittelyyn. Proteiinitransduktion toimivuus osoitettiin myös in vivo= –koesarjassa, jossa I8 PpoIvektorikäsittely= hidasti tuumorien kasvua. Meganukleaasin spesifisyys= kohdealueiden suhteen todettiin kromatiiniimmunopresipitaation ja genominlaajuisen syväsekvensoinnin avulla. Villityypin integraasin ja kromatiinin interaktioita= kartoitettaessa havaittiin virusbiologisesti mielenkiintoisia= eroja interaktio= ja integraatiojakauman välillä. Kokonaisuutena työ osoitti suoran= pakkausmenetelmän käyttökelpoisuudenproteiinitransduktioonsekäintegraationkohdentamiseen. = Luokitus:QU470,QZ52,QW51,QW168.5.R18,QW160
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