Generation of HLA universal platelets for regenerative applications

Constança Figueiredo

Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany

IMGT/HLA database

12.672 HLA alleles

A mean of 450 new alleles/ Year

HLA loci are the most polymorphic of the entire human genome

Structure of HLA class I and II molecules

HLA-AHLA-BHLA-C

HLA-DRHLA-DPHLA-DQ

HLA Class I HLA Class II

The function of HLA molecules

Antigen presentation

Efficient immune response against pathogens and cancer cells

HLA Antigens = Major Antigens

Non-HLA Antigens = Minor Antigens = Presented Peptides

HLA variability is the major hurdle for transplantation

MHC/non-MHCMismatch

Immune response(humoral/cellular)

Rejection

Suppression of the recipient’s immune system

Standard Strategy to prevent rejection:Immunosuppression

Control HLA expression in a allele-, gene- or class-specific way.

In a clinical setting, this would allow to

1. develop tissues with low or no immunogenicity.

2. decrease the risk of HLA-mediated post-transplanttissue/organ failure.

Aim

Universal cells and tissues

HLA TCR

Tissue(invisible)

CompetentImmune System

Reducing the organ’s Immunogenicity

Alternative Strategy to prevent rejection:Reducing the organ‘s immunogenicity

Gene silencing by RNA interference (RNAi):Small interfering RNA (siRNA)

Synthetic siRNA duplex

siRNA duplex

Cellular kinase

Single-stranded siRNA in RNA-induced silencing complex (RISC)

Translational Block Mechanism Cleavage Mechanism

mRNA mRNA

Andrew FireStanford University

Craig MelloUniversity of Massachusetts

Nobel Prize 2006

Silencing of β2-microglobulin in rat Lew fibroblasts

Figueiredo et al. BioMed Res Int, 2013

MHC silencing prolongs graft survival after allogeneic transplantation

Figueiredo et al. BioMed Res Int, 2013

Role of platelets

Maintain Haemostasis

Secretory functions

Modulation of immune responses

Regulate proliferation

Application of platelets in regenerative medicine

Treatment of thrombocytopenia

Wound healing

Tissue remodelling

Drug carriers

Thrombocytopenia

Characteristics

• Low platelet number

• Major bleeding complications

Ex-vivo induced megakaryocyte

progenitors

TPO receptor agonistsAllogeneic platelets

Alloimmunization against HLA class I

antigens

Platelet refractoriness

due to HLA antibodies

HLA-universal platelets

Treatment options

In vitro production of Platelets to treat platelet transfusion refractoriness

Thrombopoiesis

MEP

Thrombopoietin (TPO)

Stem cell factor (SCF), IL-3IL-6, IL-11

CFU-MK Promegakaryoblast

Cell division

Megakaryocyte

Proplatelet producing megakaryocyte

Platelets

CD41 (GPIIb/IIIa)

CD42a (GPIX), CD42b (GPIb)

Endomitosis

T CAAGAG

5′- GAATGGAGAGAGAATTGAA3′- TTCTTACCTCTCTCTTAACTT

T

A

HLA class I-specific silencing

H1 shRNA EF1a GFP

Silencing HLA expression

Figueiredo et al, J mol Med, 2006Figueiredo et al, Transfusion, 2007Figueiredo et al, BioMed Res Int, 2013Wiegmann & Figueiredo et al, Biomaterials, 2014

Polyploidy of differentiated megakaryocytes

PBMCs CD41+ shRNA_NS CD41+ shRNA_β2m

35% 33%0,1%

Cel

l co

un

t

PI

2n4n

8n

16n

HLA class I silencing

64%

shNS shβ2mNC

white bar = 50μm

CD

42a

shNS shβ2m

CD61

isotype

29.5 % 33.7 %0.0 %

ProPLTs

PLTs

Cel

l co

un

t

PAC-1

shNS + ADP shβ2m + ADP shNS

25.1% 29.4%2.0%

(n = 4)

Gras & Figueiredo et al. Hum Gen Ther, 2013

ProPLT/ PLT-production by differentiated MKs

HLA-universal MKs protected from antibody-mediated cytotoxicity

sh

β2

ms

hN

S

NC PC aHLA03

(n = 4, ***p<0.001)

HLA-universal MKs & PLTs prevent PLT refractoriness in a mouse model

NSG NSG

antiHLA ab

shNS shβ2mNC (ab only) ab + shNS ab + shβ2m

HLA-universal PLTs prevent PLT refractoriness in a mouse model

huCD61

huC

D42

a

shNS shβ2mIsotype control

NC (ab only) ab + shNS ab + shβ2m

Human PLTs in the circulation of NSG mice

(n = 3, *p<0.05, **p<0.01)

Gras & Figueiredo et al. Hum Gen Ther, 2013

Induced pluripotent Stem (iPS) cells as unilimited cell sources

Yamanaka S. Nature Medicine 2009.

Differentiation of iPSCs toward HLA-universal PLTs

iPSC colony Silencing HLA expression iPSC cultured in monolayer

Orbital shaker Megakaryocyte andPlatelet formation

BMP-4 VEGF

TPO

Mesoderm

Megakaryocytes

Hematopoiesis/MK

SCFIL-3TPO

Xenofree conditions

Generation of HLA-universal iPSC lines

SSEA4

Tra-1-60Before

Td P1 P5

P10 P150

50

100

150shNSshß2m

% H

LA

exp

ress

ion

shNS shß2m

Monolayer in Laminin

NC shβ2m

Co

un

tsC

ou

nts

CD41

isotype day 12

day 19 day 26

0.2 % 7.3 %

82.1 % 44.6 %

Generation of HLA-universal Megakaryocytes

shNS

shß2m

0

20

40

60

80

100

% H

LA

exp

ress

ion

MKs

Differentiation of Platelets

CD61

shβ2mNS

13.4 %18.6 %

CD

42a

CD62P

Co

un

tsNon-stimulated ADP + Thrombin

iPSC-derived Platelets respond to external stimuli

MKsmembrane

PLTs

23.8 %

Large-scale production of platelets in bioreactors

CD61

Summary

• The generation of in vitro pharmed genetically modified platelets is feasible.

• IPSC serve as an unlimited cell source for the large-scale production of platelets.

• Universal platelets bring the concept of Universal cells one step closer to reality.

Institute for Transfusion Medicine, Hannover Medical School, Germany.Rainer Blasczyk Dominca RatusznyLaura Schlahsa Haijiao ZhangAnn-Kathrin Börger Stefanie VahlsingLilia Goudeva

Helmholtz Center for Infection Research, Braunschweig, GermanyCarlos GuzmanKai Schulze

Institute of Experimental Hematology, Hannover Medical School, HannoverThomas Moritz, Axel Schambach Nico LachmannMania Ackermann

LEBAO, Hannover Medical School, HannoverUlrich MartinStefanie WunderlichLena Engels

Acknowledgements

Stiftung für Transfusionsmedizin