PD-L1gene*coverexpression:anewstrategyforType1Diabetes

Mellitus

AndreaGaleone,AntoninoLaRocca,ViDoriaPereE,RebeccaRosseE

A.A.2017/2018Prof.ssaIsabellaSaggio

(Chenetal.,2017MolecularMetabolism)

Introduc*on

ClinicalfeaturesT1DM•  Autoimmunereac;onagainstpancrea;cβ-cells•  Lackofinsulinproduc;onandhyperglicemia•  Attheonsetofthedisease,30-40%ofpancrea;c

isletsarealive

Currenttreatments•  Frequentmonitoringofbloodglucoselevelsand

synthe;cinsulinadministra;ons•  Clinicalpancrea;cislettransplanta;on

T1DMandcogni*vedysfunc*on

•  Slowingofmentalspeed•  Adiminishedmentalflexibility•  Lerningandmemoryarespared

(AllisonL.O’Kelletal.,2017Diabetes)

Non-obesediabe*cmice(NOD)

Why?StrikingresemblancetohumanT1DM:•  Pathophysiology•  Diseasedevelopment•  Autoimmunerejec*onofislettransplants•  MHCclass2sharestructuralsimilari;es

(Kingetal.,2012Bri;shJournalofPharmacology)(AllisonL.O’Kelletal.,2017Diabetes)

WT

NOD

PD-L1/PD-1pathway

(NataliaMar;n-Orozcoetal.,2006JEM)

IsletcellsprotectthemselvesbyexpressingPD-L1.BindingofPD-L1toPD-1receptordownregulates

T-celleffectorfunc*on.

PD-L1 PD-1

(BenNasretal.,2017ScienceTransla;onalMedicine)

PD-L1KOingra\edisletsfavoredimmunecellinfiltra*onanddecreasedtheisletfunc*on.

Published:March18,2016

PD-L1KOdoesn’tshowimpairmentofinsulinerelease.

Objec*ve

Experimentalplan

RestoretheexpressionofPD-L1inpancrea*cisletstoblocktheautoimmunereac*on,usingamodified 𝟑↑𝒓𝒅 genera*onLen*viralvector.

Laparotomyandpancreasmobiliza*on Len*viralinjec*on PD-L1overexpression

𝟑↑𝒓𝒅 genera*onLen*virus

CMV R U5 ψ/RRE CMVpromoter CD274 GFP WPREcPPT R U5

5’LTRwithnoU3 SIN/3’LTR

238bp290bp508bp

Ad-GFP

LV-GFP

Advantage:•  Longtermgeneexpressionviastablevector

integra;onintohostgenome;•  Capacityofinfec;ngbothdividingandnon-dividing

cells;•  Lackofimmunogenicviralproteinsaiervector

transduc;on;•  Rela;velyeasysystemforvectormanipula;onand

produc;on.

(GiannoukakisN.etal.,1996GeneTher)

Vectorbuilding–Len*virus

CMV Rev poliACMV Gag Pol poliARRE

CMV LCMV poliA

CMV RU5 ψ/RRE CMVpromoter CD274 GFP WPREcPPT R U5

5’LTRwithnoU3 SIN/3’LTR

Envelopevector

Packagingvectors

Transfervector

VSV-G LCMV

Transfervector Packagingvectors

Envelopevector

Collectsupernatanta\er24-48h

(Kobinger,2004HumGeneTher.)

Transduc*oninvitro

WT

NOD+LV-PD-L1

NOD

WT+LV-PD-L1

PD-L1expression

WB

Real-*mequan*ta*vepolymerasechainreac*on(PCR)

PD-L1

β-ac*n

WT NOD NOD+LV-PD-L1

IsletsinfectedwithLV-PD-L1athighdoseshowPD-L1overexpressionandnotoxicity.

%GFP-posi*vecells

FACSWT

LV-GFP

1X

𝟏𝟎↑𝟓 

pfuLV

-GFP

2X

𝟏𝟎↑𝟔 

pfu

Lowdose Highdose

BrainSkinLungHeartMuscle

LiverKidneyPancreas

Tgfreq

uencyin*ssue

s(%)

Transduc*oninvivo

ELISA

WT

NOD+LV-PD-L1

NOD

0

40

20

60

100

Insulin

erelease

(ng/mgprotein)

PD-L1expressionblocktheautoimmunereac*on,avoidingtheprogressivedistruc*onofpancrea*cislets.

%lossofp

ancrea*cIslet

Week3

Week9

Week14

WTNODNOD+LV-PD-L1

100

50

NODmiceweretransducted5daysbeforetheanalysis.

NOD NOD+LV-PD-L1

Infiltra*

on(%

isleta

rea)

Diabetes(weeks)

0 200

Tgfreq

uencyinPan

crea*cislets(%

)

Daysa\erinjec*onLV-GFP20

0.1

100

1

10

WeexpectstableLV-GFPexpressionformorethan200days.

40510 100

30

60

n=45

n=10

n=25

Pimallsandsolu*ons

InthehumanT1DMisimpossibletoknowtheaccuraterateoflossofpancrea;cislets.Unfortunately,there’snodiagnos;cwaytodetectdiabetesintheearlystageofthediseaseandthisisthemainobstacletoourproject.Hopefully,infuturewecouldtreatdiabetesbeforetheonsetofthehyperglicemiaandsosavemoreisletsdirectinthepa;ents’pancreas,avoidingtransplants.Moreover,isletsareonlyasmallfrac;onoftheen;repancreasandthesurgeryshowsahighpercentualofallograirejec;on.Inthefuture,itmightbepossibletoworkdirectlyonhumanpancreas,inordertorestorethePD-L1expressionandavoidautoimmunereac;on.

Costand*me

InvitroexperimentsLen*vectorconstruc*on Invivoexperiments Results

1year 2year

30WTmiceC57BL/6J≈€53085NODmice≈€2.787Len;vectorproduc;on≈€1500Westernblotkit≈€2000RT-PCRkit≈€500A293Cells100µg:€690Ultrasensi;vemouseInsulinELISAkit:€320PD-L1an;body3000µl:€300Stabula;on:€25.000/years

Intotal≈€35.000(withoutsalaryofresearchers)

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