Treatment of type 1 diabetes via α-cellinsulin production · 2019. 3. 28. · Treatment of type 1...
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Treatment of type 1 diabetes via α - cell insulin production Bertozzi Alessia Buccioli Lucia Tosato Federica Gene Therapy Professor Isabella Saggio A.Y. 2018/2019
Transcript of Treatment of type 1 diabetes via α-cellinsulin production · 2019. 3. 28. · Treatment of type 1...
Treatmentoftype1diabetesviaα-cell insulinproduction
BertozziAlessiaBuccioli LuciaTosatoFederica
GeneTherapyProfessorIsabellaSaggioA.Y.2018/2019
Backgroundoftype 1diabetes• Autoimmunedisease causing pancreatic β-cell
ablation• Insulin deficiency leading tohyperglycemia
BluestoneJ.A.etal.Nature(2010)
Ourgoal
Findapossibletherapeuticapproachinhuman
How?
InducinginsulinproductionandPdx1overexpressioninpancreaticα-cells
Cigliola V.etal.Nature (2018)
Ourmodels
NonObeseDiabeticMouse
SpontaneousT1Dmodel
Humanpancreaticorganoid
Cell-derivedinvitro3Dorganmodel
Loomans C.J.M.etal.StemCellReports(2018)
Invivotherapeuticapproach
LiaoH.etal.Cell(2017)
Injectionof:• AAV8-Cas9• AAV8-dgIns1-MPH• AAV8-dgPdx1-MPH
+
Invitrotherapeuticapproach
Humanpancreaticorganoid culture
Transfectionof:• AAV8-Cas9• AAV8-dgIns-MPH• AAV8-dgPdx1-MPH
+
Deliverysystemof gRNAs
AAV8sgRNA Ins1(5’->3’)1 GGGACATCAATATT
2 TCTACTTAGTCTTA
3 TATAGTCTTAATAA
4 ACCTTCTTCATCTT
sgRNA Pdx1(5’->3’)
1 TGTGCGCCCCGTTT
2 AAGCCTCCTTCTTA
3 GCCGTGCCATAGGC
4 GTGCAGGTGTTCGC
sgRNA Ins (5’->3’)
1 GCTGAGGCTGCAAT
2 GCACCAGGGAAATG
3 ATGACCCGCTGGTC
4 TCTGGCCACCGGGC
sgRNA Pdx1(5’->3’)
1 GTGCTCCCCAAAAT
2 AAGAGGCTAGGCCC
3 GGCCGCCGCACCAT
4 CGCACTAAGAGGCT
CHOPCHOP
Delivery system
dgRNA ITRPolyAMPHCAGITR U6
ITR ITRPolyANLSNLSCMV Cas9
Cas9
AAV8
dgRNA
AAV8Addgene
Predictiveresults
14bp-modifieddgRNA improvesLuciferasegeneactivationinα cells.
ExpressionofCas9inbothinjectedNODmouseandtransfectedhumanorganoid.
AdaptedfromLiaoH.etal.Cell (2017)
PredictiveresultsInvivo
Immunofluorescenceofbihormonalglucagon-insulinα-cells.
AdaptedfromCigliola V.etal.Nature(2018)
Glucose-responsiveinsulinproduction.
PredictiveresultsInvivo
ImmunofluorescentanalysisofPDX1proteinlevelinpancreastissue.
AdaptedfromLiaoH.etal.Cell (2017)
Pdx1geneinductionanalysis(qRT-PCR).
PredictiveresultsInvitro
InsgeneexpressioninhumanpancreaticorganoidbyRT-PCR.
AdaptedfromGimènez C.A.etal.GeneTherapy(2016) AdaptedfromZhaoC.etal.Moleculartherapy(2015)
Immunofluorescenceofinsulinandglucagoninhumanpancreaticorganoid.
PredictiveresultsInvitro
ImmunofluorescenceofPDX1inhumanpancreaticorganoid.
AdaptedfromKuboA.etal.PLoS One(2011)
Detection ofPDX1level.
LongtermpredictionsInvivo
AdaptedfromRecino A.etal.Nature(2018)
Pitfallsandpossiblesolutions
Ø Cas9off-targets
Ø AAVspecificity
Ø Restoration ofautoimmuneresponseagainst insulin-producingα cells
newCas9types with enhancedspecificity (eSpCas9)
Y447F+Y733F mutantsofAAV8
CostsandtimeNod mice(x40) 1.550€ (Jackson laboratory)Stabulation formice about440€/monthPancreatic organoids 1.400€Cas9vector 57€each(Addgene)dgRNA vector 57€each(Addgene)AAV8(10^12GC/mLx20mL) 10.000€(Addgene)Immunofluorescence kit 516€(ThermoFisher)Westernblotkit about1500€Immunohistochemistry antibodies about300€xAb+ secondaryAbsqRT-PCRkit 690€(Sigma-Aldrich)Luciferase 300€MolecularBiologyLabApparatus 500€
1styear 2ndyear 3rdyear 4thyear RESULTS
References• Addgene• BluestoneJ.A.etal.Genetics,pathogenesisandclinicalinterventionsintype1diabetes.(2010)Nature.
• Cigliola V.,Ghila L.,Thorel F.,VanGurp L.,Baronnier D.,Oropeza D.,GuptaS.,Miyatsuka T.,Kaneto H.,MagnusonM.A.etal.(2018).Pancreaticislet-autonomousinsulinandsmoothened-mediatedsignallingmodulateidentitychangesofglucagon+ α-cells.NatureCellbiology.
• ChenM.,Maeng K.,Nawab A.,FrancoisR.A.etal.(2017). EfficientGeneDeliveryandExpressioninPancreasandPancreaticTumorsbyCapsid-OptimizedAAV8Vectors.HumanGeneTherapyMethods.
• ChopChop• Giménez C.A.,Ielpi M.,Mutto A.,Grosembacher L.,Argibay P.andPereyra-BonnetF.(2016).CRISPR-onsystemfortheactivationoftheendogenoushumanINSgene.GeneTherapy.
• Gromada J.,Chabosseau P.andRutterG.A.(2018).Theα-cell indiabetes mellitus.NatureReviewsEndocrinology.
• LiaoH.,Hatanaka F,Araoka T.,LuL.,RodriguezEstebanC.,Izpisua BelmonteJ.C.(2017).InVivoTargetGeneActivationviaCRISPR/Cas9-MediatedTrans-epigeneticModulation.Cell.
• JacobsonE.F.andTzanakakisE.S.(2017).Humanpluripotentstemcelldifferentiationtofunctionalpancreaticcellsfordiabetestherapies:innovations,challengesandfuturedirections.JournalofBiologicalEngineering.
• KachapatiK.,AdamsD.,BednarK.andRidgwayW.M.(2012).Thenon-obesediabetic(NOD)mouseasamodelofhumantype1diabetes.MethodsMolecularBiology.
• KuboA.,StullR.,TakeuchiM.etal.(2011).Pdx1andNgn3overexpressionenhancespancreaticdifferentiationofmouseEScell-derivedendodermpopulation.PLoSOne.
• LaperrousazB.,PorteS.,GerbaudS.,HarmaV.(2018).DirecttransfectionofclonalorganoidsinMatrigelmicrobeads:apromisingapproachtowardorganoid-basedgeneticscreens.NucleicAcidResearch.
• RecinoA.,SawyerY.,TrendellJ.,HolmesN.,WallbergM.etal.(2018).Immunosuppressionovercomesinsulin- andvector-specificimmuneresponsesthatlimitefficacyofAAV2/8-mediatedinsulingenetherapy inNODmice. Nature.
References
