M.Kissopoulos,D.Lovecchio,A.Olivieri,M.Or5cello,C.Sferra

GENETHERAPYCOURSE2017/2018Prof.ssaIsabellaSaggio

T1DMandImmunesystemevasion:Evalua5onofastemcell-basedapproach

Type 1 diabetes mellitus

•  Autoimmunediseasethatdevelopsasaconsequenceof:

-Gene5cpredisposi5on

-Environmentalfactors

-  Stochas5cevents

•  Characterizedbyanorgan-specificimmunedestruc5onofinsulin-producingβ-cells

ABSOLUTEINSULINDEFICIENCY

•Itisassociatedwithanincreasedriskof

heartdisease,stroke,blindness,kidneyfailure.

•  Thereiss5llnotacure,hyperglycemiaistakenunder

controlbydailyinsulinadministra5on.

J.A. Bluestone et al.,Nature (2010)

IPSCells

Len+viralInfec+on

•  IDO•  PDL1+CTLA4•  PDL1+CTLA4+IDO

C-IPSCellsβ-likecells

Differen5a5oninPancrea5cβ-likecells:

«Lineage-controlNetwork»protocol

De-differen5a5onthroughYamanaka’s

factors

Cellsinjec5onInKidneyandLiver

NephrectomyHepatectomy

Invivoresults

NODmouse

Goals:•  Replacementoffunc5onalβ-like cells

•  Autoimmuneevasion

1

2

34

Overviewofourstrategy

NODmouse•  TheNon-ObeseDiabe+cmousemodelmatchescloselyenoughwithT1DMpa+ents

J.A.Pearson et al., J.Autoimmun. (2016)

•  Glycosuria•  Polyuria•  Weightloss

•  Lymphocy5cinfiltra5oninLangheransislets

•  Highincreaseofglucoselevelsinbloodstream

•  Noinsulinproduc5on

•  Destruc5onofpancrea5cβcells

TYPE1DIABETES

•  Skeletricandcardiacmusclesproblems

MAF-derivediPScellsandvalida5onofpluripotency

Soma5ccells:MAF(mouseAdultFibroblast)

NODmouse

NOD-iPScells

Yamanaka’sfactorsreprogramming

K.Takahashi et al., Cell (2006) te

ratomafo

rma5

on

Func5onalinvivocontrol

CellularandmolecularinvitrocontrolsWTNOD-iPSC

1

Neomicinselec5on

WhatarewegoingtodowithMAF-derivediPScells?NODmouse

NOD-iPScells

NOD-iPScellscorrected

Viraltransduc5on

NOD-iPScellsnot-corrected

TcellAn5genpresen5gcell

www.adipogen.com

HowtocorrectiPSC?Bylen+viralvectormediatedgenetherapy:

Polycystronictransferplasmidwithacons5tu5vepromotercontainingthetransgenesseparatedbyaself-cleavableT2Apep5delinker:1.  IDO2.  PD-L1+CTLA43.  PD-L1+CTLA4+IDO

Vectortakenfromwww.Addgene.org

2

1.

2.

3.

M.B.Nasretal.ScienceTransla7onalMedicine(2017)

Trasduc5onefficiency

•  FACSanalysis:

•  Proteicexpressionevalua5onthroughWesternBlotanalysis:1.  Mock-transducedcells2.  Untreatedcells3.  IDO4.  PDL-1+CTLA45.  PDL-1+CTLA4+IDO

goodtransduc5onEfficiency.

Whyalen5viralvector?

Manyadvantages:•  strongandcons5tu5veexpression•  long-termefficacy•  containsupto7,5-8,5kb•  Randominser5onsiteslowerriskofgenotoxicityseenbyPCRandsequencingoftheamplicon

TOXICITYEVALUATION:Cellviabilityanalysis:highpercentageofviablecellsobtained.

Safetyevalua5on

LV genomic integration Profile

Alessandra Biffi et al., Science 341 (2013)

Common insertion site analysis.

Alessandra Biffi et alScience 341.(2013)

«Lineage-controlnetwork»protocol3

P.Saxena et al., Nature Communication (2016)

«Lineage-controlnetwork»protocol

P.Saxena et al., Nature Communication (2016)

AretheiPSC-derivedβ-likecorrectedcellsgoodenoughinvitro?

P.Saxena et al., Nature Communication (2016)

qPCR Glucose response analysis

Insulin secretion analysis

β-likeCorrectedcell

EndocrineProgenitorcell

MeasurementusingELISAtest.

•  Invitroisshownagoodtranscrip5onprofileforthepancrea5nkeygenes

•  Invitrothelineage-controlnetwork-derivedβ-likecellsareinsulin-secre5ngandglucoseresponsive

InvivoanalysisdemonstratethattheiPSCnetwork-derivedβ-likecells:•  showthetypicalinsulin-storagevesciclesthatarefoundinmaturepancrea5cbetacells.

1µm 2µmTrasmission-electron

micrographs

AretheiPSC-derivedβ-likecorrectedcellsgoodenoughinvivo?

P.Saxena et al., Nature Communication (2016)

β-likecellsNC β-likecellsLV-IDO

β-likecellsLV-PDL1-CTLA4Ig β-likecellsLV-PDL1-CTLA4Ig-IDO

1.Tcells2.βcells

0102030405060708090

**

**

Cellsvita

lity

**

Couldourβ-likecorrectedcellsescapetheimmunesystem?

W1 W2 W3 W4 W5 W6 W7 W8 W9 W10 W11 W12 W13 W14 W15

W1 W2 W3 W4 W5 W7 W8 W9 W10W6

Nephrectomy

Hepatectomy

X15

NODmice5weeksold

NODmice5weeksold

InjectedcellKidney

InjectedcellLiver

Bloodglucoselevelevery4days

ImmunohistochemistryAnalysisforInsulinandC-Pep5de

Sameexperimentsinparallelhavebeendonewithinjec5onofdifferen5atedbetalikecellswithoutcorrec5on.

Invivoexperiments4

UsingLV-PDL1-CTLA4-IDOexpressionweobtainedglucoselevelswithinsafetyrangeresemblingtoWT

LV-IDO LV-PDL1+CTLA4Ig

Fas5ngblood

glucoselevel(mmol/L)

LV-PDL1+CTLA4Ig+IDO

Fas5ngblood

glucoselevel(mmol/L)

Averagebloodglucoseandcircula5nginsulinlevels

0

0.2

0.4

0.6

0.8

1

1.2

Fas5ngCircula5

nginsulinlevel(ng/m

l)

Circula5nginsulin

MeasurementusingELISAtest.

**

**

**

0

5

10

15

20

25

0 10 15weeks

Averagemerged

IDO PDL-1+CTLA4 PDL1-CTLA4+IDO

UntreatedNOD WT

Fas5ngblood

glucoselevel(mmol/L)

NephrectomyHepatectomy

Invivoimmunestaining

β-likecellsC-pep5deInKidney.

NODmouse WT

IDO Pdl-1+CTLA-4

β-likecellsInsulininkidney.

Nodmouse WT

IDO Pdl-1+CTLA-4

Weobtainedthesameresultsfromβ-likecellsintheliver

β-likecellsleukocytecommonan5genexpressioninKidney

an5-C-pep

5dean5

-C-pep

5dean5

-C-pep

5de

an5-insulinan5-insulinan5

-insulin

an5-CD4

5an5

-CD4

5

Pipallsandameliora5ons

•  Inducedpluripotentstemcellsstability,efficiencyandsafetycouldbeamelioratedusingmiRNAsinsteadofYamanaka’sfactorsorusingNanogandLin28insteadofc-Myc

•  NODmousecanbehumanized.

•  Accordingtoourstudieswesuggesttogoaheadwithexperimanta5oninnon-humanPrimates.

•  Challengess5llremainfornon-humanPrimatesβ-likecellsdifferen5a5on.

•  Suicidegenescouldbeawaytoenhancethesafetyofexvivogenetherapy,byelimina5ngthetransducedcellsatthesiteofimplanta5on.

F.Alaeeetal.,2014GeneTherapy

Costs and Time•  WT mouse+ NOD mouse: 26$ (x51 WT mouse) + 44$ (x50 NOD mouse) •  Stabulation for the mice: about 500$/month •  Culture dishes (Sigma-Aldrich): 119$ •  Yamanaka’s factors plasmid: 65$ •  Lipofectamine LT Reagent with Plus Reagent (Invitrogen) 0,75ml: 400€ •  qPCR (miScript SYBR Green PCR Kit- QIAGEN): 451$ •  Toxicity assay: 400$ •  Taq PCR Core kit (QIAGEN): 171$ •  Lentivirus (1ml at titer >1x10^6 TU/ml) and plasmid (Addgene): 250$ (x5) •  Next Generation Sequencing: 1500-3000€ •  FACS antibodies: 200-300$/each + respective controls •  Immunohistochemistry antibodies: 200-300$/antibody + secondary antibody •  ELISA assay kit (biorbyt): 580$/plate •  Western blot antibodies: 300-400$/antibody + secondary antibody •  Supplementary costs including routine lab experiments are not evaluable

Timeofwork:5years,70000/80000 $

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•  www.addgene.org