New therapies : EU-supported research in genomics and biotechnology for health : sixth framework programme (2002-2006).
Date: 2008-01-25
Institutions: Directorate-General for Research and Innovation
Subjects: biotechnology, catalogue, medical research, research programme, therapeutics

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New Therapies
Modern  biomedical  research  is  opening  up  many  novel  approaches  to  therapy. These 
include gene and cell therapy, immunotherapy, tissue engineering and regenerative med-
icine. These approaches offer hope for therapy of diseases which are currently untreat-
able, where life is at stake, and for regenerating diseased, damaged or defective tissues 
and organs. This is a very active research area in Europe, involving scientists, clinicians and 
industry, as well as patient organisations, ethical committees and regulatory authorities. 
Research in new therapies formed part of the EU’s Sixth Framework Programme (2002-
2006) and the purpose of this catalogue is to demonstrate the activities initiated over the 
duration of the programme and the initial results obtained.

Price (excluding VAT) in Luxembourg: 25.00 EUR  

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Interested in European research? 
Research*eu is our quarterly magazine keeping you in touch with main developments (results, pro-
grammes, events, etc.).  
It is available in English, French and German. A free sample copy or free subscription can be obtained 
from:

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Internet: http://europa.eu.int/comm/research/rtdinfo/index_en.html

EUROPEAN COMMISSION

Directorate F – Health

Unit F5 – Health Biotechnology

Contact: Charles Kessler

Office CDMA 2/188

Tel (32-2) 29 56112

Fax (32-2) 29 94693

E-mail: charles.kessler@ec.europa.eu 

Directorate F – Health

Unit F5 – Health Biotechnology

Head of Unit 

Arnd HOEVELER (arnd.hoeveler@ec.europa.eu)

Secretary: Andrea JAEGER (andrea.jaeger@ec.europa.eu)

New Therapies  Charles KESSLER (charles.kessler@ec.europa.eu)

Fatiha SADALLAH (fatiha.sadallah@ec.europa.eu)

Juergen SAUTTER(juergen.sautter@ec.europa.eu)

Secretary: Caroline RICHMOND (caroline.richmond@ec.europa.eu)

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EUROPEAN COMMISSION

NEW THERAPIES

EU-supported Research in Genomics and Biotechnology for Health
Sixth Framework Programme (2002-2006)

Edited by
Charles Kessler

2007

Life Sciences, Genomics and Biotechnology for Health

EUR 22841

Directorate-General for Research

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NEW THERAPIES

Europe Direct is a service to help you find answers  
to your questions about the European Union 

Freephone number (*): 
00 800 6 7 8 9 10 11 

(*) Certain mobile telephone operators do not allow access to 00 800 numbers or these calls 

may be billed. 

More information on the European Union is available on the Internet (http://europa.eu). 

Cataloguing data can be found at the end of this publication. 

Luxembourg: Office for Official Publications of the European Communities, 2007 

ISBN 978-92-79-05562-1 

© European Communities, 2007 
Reproduction is authorised provided the source is acknowledged. 

Printed in Belgium 

PRINTED ON WHITE CHLORINE-FREE PAPER 

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NEW THERAPIES
NEW THERAPIES

TABLE OF CONTENTS

INTROdUCTION

REgENERATIvE
MEdICINE

11

1

EuroStemCell
European consortium for stem cell research

17

gENOSTEM
Adult  mesenchymal  stem  cells  engineering  for 
connective  tissue  disorders.  From  the  bench  to 
22
the bed side

OsteoCord
Bone from blood: optimised isolation, characteri-
sation  and  osteogenic  induction  of  mesenchy-
27
mal stem cells from umbilical cord blood

TherCord
Development  and  preclinical  testing  of  cord 
30
blood-derived cell therapy products

EPISTEM
Role  of  p63  and  related  pathways  in  epithelial 
stem cell proliferation and differentiation and in 
32
rare EEC-related syndromes.

Ulcer Therapy
Gene  transfer  in  skin  equivalnts  and  stem  cells: 
novel strategies for chronic ulcer repair and tis-
37
sue regeneration

Skintherapy
Gene therapy for Epidermolysis Bullosa: a model 
system for treatment of inherited skin diseases.


41

THERAPEUSKIN
Ex vivo gene therapy for recessive dystrophic epi-
dermlysis bullosa : preclinical and clinical studies
44


BetaCellTherapy
Beta cell programming for treatment of diabetes
47


EuroSTEC
Soft  tissue  engineering  for  congenital  birth 
defects  in  children:  new  treatment  modalities 
for  spina  bifida,  urogenital  and  abdominal  wall 
2
defects

SC&CR
Application and process optimisation of human 
7
stem cells for myocardium reapair

STEMSTROKE
Towards a stem cell therapy for stroke

61

STEMS
Preclinical  evaluation  of  stem  cell  therapy  in 
64
stroke

STROKEMAP
Multipotent Adult Progenitor Cells to treat Stroke
67


RESCUE
From stem cell technology to functional restora-
69
tion after spinal cord injury

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New Therapies – Table of contents



NEW THERAPIES

TABLE OF CONTENTS

gENE THERAPY

83

STEM-Hd
Embryonic stem cells for therapy and exploration 
72
of mechanisms in Huntington Disease

CLINIgENE
European Network for the Advancement of Clini-
8
cal Gene Transfer and Therapy

NEUROscreen
The  discovery  of 
molecules

future  neuro-therapeutic 
7

myoamp
Amplification  of  human  myogenic  stem  cells  in 
77
clinical conditions

CRYSTAL
CRYo-banking of Stem cells for human Therapeu-
80
tic AppLicationp

CONSERT
Concerted Safety & Efficiency Evaluation of Ret-
roviral Transgenesis in Gene Therapy of Inherited 
92
Diseases

gIANT
Gene therapy: an Integrated Approach for Neo-
98
plastic Treatment

BACULOgENES
Baculovirus vectors for gene therapy

102

THOvLEN
Targeted  Herpesvirus-derived  Oncolytic Vectors 
104
for Liver cancer European Network

THERAdPOX
Optimised and novel oncolytic adenoviruses and 
pox viruses in the treatment of cancer: Virothera-
py combined with molecular chemotherapy107

RIgHT
RNA  Interference  Technology  as  Human  Thera-
110
peutic Tool

ZNIP
Therapeutic  in  vivo  DNA  repair  by  site-specific 
11
double-strand breaks

SNIPER
Sequence  Specific  Oligomers  for  in  vivo  DNA 
118
Repair

Improved precision
Improved precision of nucleic acid based therapy 
121
of cystic fibrosis

6

New Therapies – Table of contents

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NEW THERAPIES

InvivovectorTrain
European labcourse: towards clinical gene thera-
147
py: preclinical gene transfer assessment

IndustryvectorTrain
European  labcourse:  advanced  methods  for  in-
dustrial  production,  purification  and  characteri-
10
sation of gene vectors

INTHER
Development  and  application  of  transposons 
and  site-specific  integration  technologies  as 
non-viral gene delivery methods for ex vivo gene-
124
based therapies

Epi-vector
Episomal  vectors  as  gene  delivery  systems  for 
129
therapeutic application

PolExgene
Biocompatible non-viral polymeric gene delivery 
systems  for  the  ex vivo  treatment  of  ocular  and 
cardiovascular diseases with high unmet medical 
133
need

Magselectofection
Combined  isolation  and  stable  nonviral  trans-
fection of hematopoietic cells ‘ a novel platform 
technology  for  ex  vivo  hematopoietic  stem  cell 
13
gene therapy

Synthegenedelivery
Ex  vivo  gene  delivery  for  stem  cells  of  clinical 
interests  using  synthetic  processes  of  cellular 
and  nuclear  import  and  targeted  chromosomal 
138
integration

MOLEdA
Molecular  optimisation  of  laser/electrotransfer 
DNA  administration  into  muscle  and  skin  for 
141
gene therapy

ANgIOSKIN
DNA  electrotransfer  of  plasmids  coding  for  an-
tiangiogenic  factors  as  a  proof  of  principle  of 
non-viral gene therapy for the treatment of skin 
144
disease

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New Therapies – Table of contents

7

NEW THERAPIES

TABLE OF CONTENTS

IMMUNOTHERAPY ANd TRANSPLANTATION

13

Allostem
The development of immunotherapeutic strate-
gies to treat haematological and neoplastic dis-
eases on the basis of optimised allogeneic stem 
1
cell transplantation

dC-THERA
Dendritic cells for novel immmunotherapies


19

dC-vACC
Dendritic cells as natural adjuvants for novel vac-
16 
cine technologies

THERAvAC
Optimised delivery systems for vaccines targeted 
169
to dendritic cells

dENdRITOPHAgES
Therapeutic cancer vaccines

173

genomes To vaccines
Translating  genome  and  proteome  information 
177
into immune recognition

COMPUvAC
Ration  design  and  standardised  evaluation  of 
181
novel genetic vaccines.

HEPACIvAC
New  preventative  and  therapeutic  Hepatits  C 
186
vaccines: from preclinical to phase I

BacAbs
Assessment  of  structural  requirements  in  com-
plement-mediated bactericidal events: towards a 
global approach to the selection of new vaccine 
189
candidates

Mimovax
Alzheimer’s  disease-treatment  targeting  trun-
193
cated Aß40/42 by active immunisation

Pharma-Planta
Recombinant  pharmaceuticals  from  plants  for 
196
human health

SAgE
SME-led antibody glyco-engineering

200

BMC
Bispecific  monoclonal  antibody 
concept

technology 
202

AUTOCURE
Curing  autoimmune  disease.  A  translational  ap-
proach to autoimmune diseases in the post-ge-
nomic era using inflammatory arthritis and my-
ositis as prototypes and learning examples 204

INNOCHEM
Innovative chemokine-based therapeutic strate-
gies for autoimmunity and chronic inflammation
209


CELLAId
European symposia for the evaluation of poten-
tials  and  perspectives  of  curative  cell  therapies 
214
for autoimmune diseases

STEMdIAgNOSTICS
The  development  of  new  diagnostic  tests,  new 
tools and non-invasive methods for the preven-
tion, early diagnosis and monitoring for haemat-
217
opoietic stem cell transplantation

RISET
Reprogramming the immune system for the es-
221
tablishment of tolerance

8

New Therapies – Table of contents

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NEW THERAPIES

XENOME
for 
Engineering  of 
xenotransplantation  studies  in  primates:  a  step 
224
towards clinical application

the  porcine  genome 

CLINT
Facilitating 
trials in stem cell transplantation

international  prospective  clinical 
228

TRIE
Transplantation 
Europe

research 

integration  across 
230

INdEX OF PROJECTS

233

INdEX OF 
COORdINATORS

234

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New Therapies – Table of contents

9

NEW THERAPIES

10

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NEW THERAPIES

INTROdUCTION

“New therapies” as used in the title here 

refers to treatments such as gene and 
cell  therapy,  immunotherapy,  tissue 
engineering  and  regenerative  medicine  which 
offer hope for therapy of diseases which are cur-
rently untreatable, where life is at stake, and for 
regenerating  diseased,  damaged  or  defective 
tissues  and  organs.  Their  main  characteristic  is 
that  they  employ  large  biomolecules,  genes, 
cells and tissues as therapy rather than drugs or 
pharmaceuticals. By addressing such conditions 
as  arthritis,  diabetes,  heart  and  neurodegenera-
tive disease, new therapies address problems of 
an  ageing  population,  and  as  high-value,  new 
technologies, they represent an opportunity for 
developing industrial competitiveness.

The  purpose  of  this  compilation  of  information 
on  EU-sponsored  research  in  new  therapies  is 
to  demonstrate  the  range  of  activities  under-
taken  in  this  field  during  the  course  of  the  EU’s 
Sixth Framework Programme for Research (2002-
2006),  notably  under  the  heading “Applications 
of  knowledge  and  technologies  in  the  field  of 
genomics  and  biotechnology  for  health”  in  the 
“Life  sciences,  genomics  and  biotechnology  for 
health” thematic priority.

The  projects  described  have  been  ordered  as 
much  as  possible  on  the  basis  of  similarity  and 
have been grouped into three chapters:

REgENERATIvE MEdICINE. This refers to innova-
tive medical therapies enabling the body to re-
pair,  replace,  restore  and  regenerate  damaged 
or diseased cells, tissues and organs. The field in-
cludes research areas such as cell therapy, tissue 
engineering,  biomaterials  engineering,  growth 

factors  and  transplantation  science.  Projects 
described here focus on regeneration of tissues 
such  as  bone  and  cartilage,  skin,  muscle,  heart, 
pancreas and nervous tissue, through the use of 
haematopoietic,  mesenchymal,  cord  blood,  hu-
man embryonic and adult stem cells. In many cas-
es projects take a comparative approach to iden-
tify the best sources of cells and to see how they 
perform  in  different  tissues.  Such  comparisons 
can  also  reveal  insights  into  underlying  knowl-
edge of the biological processes involved. High-
lights of the research include using stem cells to 
reverse  muscular  dystrophy  in  a  dog  model  of 
the disease, and developing a technique to grow 
pure brain stem cells.

gENE  THERAPY.  Gene  therapy  is  the  insertion 
of genes into an individual’s cells and tissues to 
treat a disease; its success depends on isolation 
of  effective  therapeutic  genes  and  on  finding 
suitable vectors to deliver them into the patient’s 
cells. The main approaches for gene delivery are 
viral,  which  often  displays  a  high  rate  of  gene 
transfer but which can be immunogenic and dif-
ficult  to  produce  on  a  large  scale,  and  nonviral, 
which  results in less effective gene transfer and 
limited  expression  periods,  but  which  has  no 
insert  size  limitation,  is  relatively  non-immuno-
genic  and  easy  to  manufacture.  Cell-based  vec-
tors  for  gene  delivery  use  a  patient’s  own  cells, 
which are transfected ex vivo and injected back 
into  the  same  patient,  where  they  are  non-im-
munogenic and recognised as self. Reports here 
focus on development of tools and technologies 
to  overcome  the  existing  limitations  and  allow 
clinical application of gene therapy.

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New Therapies – Introduction

11

NEW THERAPIES

INTROdUCTION

IMMUNOTHERAPY ANd TRANSPLANTATION. Immunotherapy aims to modulate the immune system 
to achieve a prophylactic and/or therapeutic goal by inducing, enhancing, or suppressing an immune 
response. Projects in this sector include cell therapy using stem cell transplantation, dendritic cells, 
vaccines and antibodies to specifi cally target cells, and research on autoimmune diseases. Transplan-
tation projects are also grouped here since immune suppression and tolerance are major research 
goals. Shortage of organs for transplantation is addressed through optimisation strategies, develop-
ment of xenotransplants and the regenerative medicine approach.

While these three areas do possess their own identity, they also contain parts that are interrelated; 
hence use of the term new therapies to cover them all. A summary of the main topics covered by the 
activities is shown in schematic version in the fi gure below:

EU New Therapies Research (FP6)

REGENERATIVE MEDICINE

IMMUNOTHERAPY

GENE THERAPY

Dendritic Cells

Episomal

Oligomers

siRNA

Cancer

Non viral vectors

Infectious diseases

Zn- finger

Electrotransfer

Mesenchymal
SC

Cord  Blood

hESC

Adult

hematopoietic
SC

Banking

Transplantation

Mechanisms

Rheuma

Cytokines

Autoimmune

islets

brain

spine

heart

Tissue  repair
& regeneration

skin

Viral/
Hep C

Alzheimer

Bacteria

Vaccinology

Selection

Design

Soft tissue

muscle

Ab Production

Transposon

Plasmid

Minichr
romosome

 AAV

Oncolytic

 Retro

Viral vectors

Lenti

Baculo

Engineered cells

Clinic

Safety

Standardisation

      Main topics of new therapies research projects 

described in this review

Integratedprojects 

The  aim  of  the  projects  described  here  was  to 
create  technological  platforms  for  the  develop-
ment  of  new  therapeutic  tools.  To  achieve  this 
projects were built around fi ve different funding 
schemes:

(16  projects).  These  are  the  largest  size 
projects  and  integrate  a  range  of  differ-
ent activities, such as multidisciplinary re-
search,  demonstration  and  training. They 
also permit projects to take a translational 
approach linking underlying biology and 
therapy  and  enable  scientists,  clinicians 
and other stakeholders to work together 
to achieve their deliverables.

12

New Therapies – Introduction

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NEW THERAPIES

SpecificTargetedResearchProjects

(29  projects).  These  are  smaller  projects 
which  focus  on  specific  research  issues. 
They  may  be  translational  but  are  less 
multi-disciplinary and wide-ranging than 
the Integrated Projects.

SME-SpecificTargetedResearchProjects 

(7  projects).  Targeted  Research  Projects 
designed  to  encourage  research  and  in-
novation  efforts  of  Small  and  Medium-
Sized Enterprises and where research-led 
SMEs play a leading role.

SpecificSupportActions (5 actions) for training, 
conferences or prospective studies in sup-
port of the programme.

Networks of Excellence (2  networks),  whose 
objective is to reduce fragmentation in EU 
research, structure the way it is carried out 
and strengthen its excellence.

The number of project supported in each of the 
three areas and the EC financial contribution to 
them is shown in the table below. It can be seen 
that overall 59 projects were supported with an 
EC contribution of almost €270 million; this was 
distributed  among  about  700  research  teams, 
almost 100 of which are SMEs. 

European  research  projects  are  encouraged 
to  be  open  towards  the  general  public  and  to 
engage  with  stakeholders  and  interest  groups. 
Accordingly  many  projects  organise  public 
meetings  and  dialogue  and  set  up  websites 
on  the  consortium,  the  research  and  results. 
Website  addresses  are  given  in  the  details  of 
each project and provide more and more up-to-
date information than this publication.

Area

Number of projects

Regenerative Medicine

Gene therapy

Immunotherapy 
and Transplantation

Total

19

19

21

59

EC financial contribution (million €)
80.6

77.4

107.8

265.8

Numbers of projects supported and EC financial contribution to new therapies research

It is clear from this compilation that new therapies is an extremely active area of research for European 
scientists both from the public and private sectors. Since the projects described here take a platform 
approach to tool and technology development applications are expected in a range of different prac-
tical settings. The recently launched Seventh Framework Programme and approval of the Regulation 
on advanced therapy medicinal products will act as a further stimulus for the area and open the way 
for much-needed clinical applications.

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New Therapies – Introduction

13

 
 
14

New Therapies – Regenerative Medicine

NewTherapy_02.indd   14

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NewTherapy_02.indd   15

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REgENERATIvE MEdICINE

16

New Therapies – Regenerative Medicine

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REgENERATIvE MEdICINE

EUROSTEMCELL

EuropeanConsortiumforStemCellResearch






ContractNo 
Projecttype

LSHB-CT-2003-0300
IntegratedProject

ECcontribution  e 11900000

Startingdate 1February2004
Duration 48months

Website  www.eurostemcell.org

Background and objectives:

The  EuroStemCell  consortium  aims  to  harness 
the  intrinsic  potential  of  embryonic,  foetal  and 
adult  stem  cells  for  the  continuous  generation 
of  specialised  cell  types. The  ability  to  produce 
differentiated cell populations at scale will create 
new  opportunities  for  the  assignment  of  gene 
function,  identification  and  validation  of  drug 
targets,  pharmaceutical  screening,  toxicology 
testing,  and,  in  the  longer  perspective,  for  the 
repair  of  diseased  or  damaged  tissue  by  cell 
transplantation. 

EuroStemCell  is  an  integrated  trans-European 
effort  to  develop  well-characterised  cellular  re-
sources  of  therapeutic  potential,  derived  from 
embryonic,  neural,  myogenic,  haematopoietic 
and  epithelial  stem  cells.  EuroStemCell  applies 
genomic and transgenic technologies in conjunc-
tion with state of the art in vitro and in vivo func-
tional assays, to dissect the molecular machinery 
that  governs  self-renewal  versus  differentiation. 
The  consortium  undertakes  comparative  mo-
lecular  and  functional  evaluations  of  stem  cells 
from embryonic, foetal and adult sources. Knowl-
edge thus acquired is used to achieve scaleable 
production  of  identified  and  characterised  cell 
lineages for in vitro gene and compound screen-
ing. These  cell  line  resources  are  also  subjected 
to preclinical evaluation of tissue integration and 
repair potential in animal models. 

Alongside basic and applied research, EuroStem-

Cell examines the ethical and societal impacts of 
stem cell research and exploitation. The consor-
tium develops outreach resources to engage pa-
tient groups, high school students and lay public 
in the issues that surround stem cell research and 
medicine.

Approach and methodology:

The  fundamental  research  work  of  EuroStem-
Cell  is  focused  on  stem  cell  identification,  isola-
tion,  and  in  vitro  propagation  and  differentia-
tion. This includes the generation and validation 
of  key  tools,  such  as  antibodies  and  genetically 
engineered cell lines and mice. Allied to this, the 
consortium  is  collating  microarray  expression 
profiling data on different stem cells. This foun-
dation work is accompanied by the evaluation of 
candidate stem cell resources for in vitro up-scal-
ing  and  drug  screening  potential.  Longer-term 
studies will evaluate the survival and functional 
integration  of  transplanted  cells  in  normal  and 
injured tissue. 

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New Therapies – Regenerative Medicine

17

 
 
 
EUROSTEMCELL

REgENERATIvE MEdICINE

es  between  partner  laboratories  funded,  while 
2  three-day  meetings  of  the  entire  consortium 
(more  than  100  researchers)  were  held,  to  pro-
mote  interaction  and  collaboration  between 
partner laboratories, and refine project planning.

Moreover,  EuroStemCell  arranged  a  major  Eu-
ropean  stem  cell  conference  series,  ‘Advances 
in Stem Cell Research’. The first conference took 
place in Milan in 2005, with an attendance of over 
300. The conference was successfully repeated in 
Lausanne in 2006, and will continue in Stockholm 
in 2007. The series attracts leading international 
speakers and participants from around the world, 
providing an important opportunity to highlight 
European  stem  cell  research.  The  consortium 
also organised annual summer schools on “Stem 
Cells and Regenerative Medicine” to develop and 
train the next generation of European stem cell 
scientists. Each school is attended by 54 pre-and 
post-doctoral  students,  including  clinicians  and 
regulators,  and  is  open  to  non-members  of  Eu-
roStemCell. 

A ‘Glossary for Stem Cell Biology’, was prepared: 
it  was  published  in  Nature  and  adopted  by  the 
EMBO Science & Society Programme in its report 
on  stem  cell  research.  Web-based  information 
about stem cell research was made accessible to 
a  range  of  audiences  at  www.eurostemcell.org. 
EuroStemCell  responded  individually  to  hun-
dreds  of  requests  for  information  from  media, 
patient groups and individuals, and it also issued 
13  press  releases,  and  attracted  worldwide  me-
dia coverage for key publications. 

The  project  partners  successfully  produced  an 
award-winning animated film, A Stem Cell Story, 
about stem cell research. The film has been distrib-
uted online, via iTunes, through festival and public 
screenings, and on DVD, and has been viewed by 
audiences totalling more than 10 000. Three ad-
ditional  short  films  to  provide  in-depth  resourc-
es  on  ethics,  cell  culture  and  cloning  have  been 
developed, in addition to a careers workshop for 

The overall flow of the project can be illustrated 
schematically, as shown below:
A  number  of  parallel  activities  have  been  de-
signed to support, extend and maximise the im-
pact of the project partners’ scientific research. In 
the first 3 years, EuroStemCell has carried out the 
following:

it developed a prototype stem cell da-
• 
tabase  to  encourage  data  exchange  and 
dissemination;
• 
it encouraged junior group leaders to 
take a higher profile in the project by ac-
cording  Associate  Principal  Investigator 
status to 8 individuals;
it  organised  specialist  workshops  fo-
• 
cusing  on  disease  areas,  to  bring  stem 
cell  researchers  and  clinical  practitioners 
together  and  develop  a ‘roadmap  to  the 
clinic’;
• 
it  held  a  series  of  workshops  on  the 
ethical implications of stem cell research, 
involving  scientists,  ethicists,  legislators, 
clinicians  and  patient  representatives  in 
discussions, and making outcomes widely 
available via the group’s website.

There were 25 training and workgroup exchang-

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REgENERATIvE MEdICINE

schoolchildren aged 13 and over, and a hands-on 
activity programme for younger children. 
EuroStemCell  initiated  joint  activities  and  re-
search  projects  with  the  BetaCellTherapy  Inte-
grated  project,  supported  by  ‘Bridge’  funding 
from the Juvenile Diabetes Research Foundation 
International. 

Expected outcome:

Over the four-year funding period, EuroStemCell 
has  been  contributing  to  advances  in  techno-
logical platforms, cellular resources and scientific 
knowledge in stem cell biology. This will provide 
the  basics  for  translational  research.  EuroStem-
Cell is accordingly beginning to establish criteria 
for preclinical validation and future clinical trials 
in the areas of neurological repair, muscle repair 
and  epithelial  repair.  A  major  outcome  of  the 
project  will  be  a ‘Roadmap  to  the  clinic’  for  cell 
therapy. 

EuroStemCell  research  will  also  facilitate  biop-
harmaceutical exploitation of stem cells, in part 
mediated via the SME partners. In this regard, Eu-
roStemCell partners have, to date, filed 24 patent 
applications, several of which have been licensed 
for exploitation by SME partners. EuroStemCell is 
committed to developing ethical guidelines and 
promoting public engagement with stem cell re-
search, with the aim of positively impacting pub-
lic and political opinion.

Main findings:

The consortium has now published 56 scientific 
papers directly supported by EuroStemCell. Many 
of  these  studies  have  appeared  in  the  leading 
peer-review  journals  including  Nature,  Science, 
Cell,  PLoS  Biology,  Developmental  Cell,  Genes  & 
Development,  Development,  Neuron  and  Jour-
nal of Neuroscience. 

In the area of basic stem cell biology, EuroStem-
Cell  investigators  have  published  fundamental 
observations,  challenging  dogmas  about  the 
origins and properties of myogenic, haematopoi-
etic and hair follicle stem cells (Nature 431; Genes 
Dev  19;  Cell  121;  Development  133;  Nature  Cell 
Biology 8).

Partners in Edinburgh, Milan and Bonn, working 
on stem cells for the nervous system have collab-
orated, achieving for the first time expansion of 
pure populations of neural stem (NS) cells, with 
the potential to generate the three cell types of 
the central nervous system (PLoS Biology 3; Cer-
ebral Cortex 16). 

Importantly,  these  neural  stem  cells  can  be  ob-
tained both from embryonic stem (ES) cells and 
from  in  vivo  tissue  sources. The  consortium  has 
also reported advances in directing both mouse 
and human embryonic stem cells into the nerv-
ous  system  lineage  by  manipulation  of  defined 

NewTherapy_02.indd   19

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New Therapies – Regenerative Medicine

19

REgENERATIvE MEdICINE

EUROSTEMCELL

signalling pathways (PLoS Biology 4). Other part-
ners  are  investigating  the  potential  of  resident 
stem cells in the adult brain to be harnessed for 
repair (Neuron 52).

In the muscle stem cell field, members of the con-
sortium have demonstrated the high capacity of 
freshly isolated muscle progenitor cells to repair 
damaged muscle (Science 309) and the potential 
of expanded mesangioblast cells for tissue repair 
in  a  large  animal  model  of  muscular  dystrophy 
(Nature 444).

Partners in Lausanne and London are advancing 
in the development of a large animal model for 
epidermal stem cell transplantation. 

SME  partners  are  coordinating  projects  to  im-
prove  culture  conditions  for  the  expansion  of 
various stem cell types. Several refined media for-
mulations have been commercialised, including 
ESGRO complete media for the growth of murine 
ES cells in serum free conditions, HEScGRO for the 
growth  of  human  ES  cells  in  serum  and  animal 
component free conditions, and N2B27 and RHB-
A  media  for  the  differentiation  of  human  and 
mouse stem cells into cells of the neural lineage. 
The  goal  is  to  develop  fully  defined  conditions 
for stem cell propagation for future therapeutic 
grade production.

The EuroStemCell ethics group has produced re-
ports on stem cell registers and stem cell bank-
ing  following  a  workshop  arranged  jointly  with 
the  Commission,  and  on  commercialisation  of 
stem cell research. These reports can be viewed 
at the following websites:
http://www.eurostemcell.org/Ethics/ethics_wrk-
shp2005.htm 
http://www.eurostemcell.org/Ethics/ethics_wrk-
shp2006.htm 

Major publications

Sampaolesi, M., Blot, S., D’Antona, G., Granger N., 

Tonlorenzi,  R.,  Innocenzi,  A.,  Mognol,  P., Thibaud, 
J.L., Galvez, B.G., Barthélémy, I., Perani, L., Mantero, 
S., Guttinger, M., Pansarasa, O., Rinaldi, C., Cusella 
De Angelis, M.G., Torrente, Y., Bordignon, C., Bot-
tinelli  R.,  Cossu,  G.,  ‘Mesoangioblast  stem  cells 
ameliorate  muscle  function  in  dystrophic  dogs’, 
Nature 444, 574 – 579.

Shinin,  V.,  Gayraud-Morel,  B.,  Gomès,  D.,  Tajba-
khsh, S., ‘Asymmetric division and cosegregation 
of template DNA strands in adult muscle satellite 
cells’, Nature Cell Biology 8, 677 – 682.

Lowell,  S.,  Benchoua,  A.,  Heavey,  B.,  Smith,  A.G., 
‘Notch  Promotes  Neural  Lineage  Entry  By 
Pluripotent  Embryonic  Stem  Cells’,  PLoS  Biology 
4(5): e121. 

Gustafsson,  M.V.,  Zheng,  X.,  Pereira, T.,  Gradin,  K., 
Jin  S.,  Lundkvist,  J.,  Ruas,  J.L.,  Poellinger,  L.,  Len-
dahl, U., Bondesson, M., ‘Hypoxia requires Notch 
signaling  to  maintain  the  undifferentiated  cell 
state’, Developmental Cell 9: 617-628.

Montarras, D., Morgan, J., Collins, C., Relaix, F., Zaf-
fran, S., Cumano, A., Partridge, T., Buckingham, M., 
‘Direct Isolation of Satellite Cells for Skeletal Mus-
cle Regeneration’, Science 309: 2064-2067

Conti, L., Pollard, S.M., Gorba, T., Reitano, E., Toselli, 
M.,  Biella,  G.,  Sun,  Y.,  Sanzone,  S.,  Ying,  Q.L.,  Cat-
taneo, E., Smith, A., ‘Niche-independent symmet-
rical  self-renewal  of  a  mammalian  tissue  stem 
cell’, PLoS Biology 3(9): e283

Adolfsson, J., Månsson, R., Buza-Vidas, N., Hultquist, 
A., Liuba, K., Jensen, C.T., Bryder, D., Yang, L., Borge, 
O.J., Thoren,  L.A.M.,  Anderson,  K.,  Sitnicka,  E.,  Sa-
saki, Y., Sigvardsson, M., Jacobsen, S.E.W., ‘Identifi-
cation of Flt3+ Lympho-Myeloid Stem Cells Lack-
ing Erythro-Megakaryocytic Potential: A Revised 
Road Map for Adult Blood Lineage Commitment’, 
Cell 121: 295-306.

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REgENERATIvE MEdICINE

Coordinator

AustinSmith
Wellcome Trust Centre for Stem Cell Research
 University of Cambridge
Tennis Court Road
Cambridge CB2 1QR, UK
E-mail: ags39@cscr.cam.ac.uk

Partners

TimAllsopp
Stem Cell Sciences UK 
Cambridge, UK

ErnestArenas,JonasFrisen,UrbanLendhal
Karolinska Institute
Stockholm, Sweden

YannBarrandon
Swiss Federal Institute for Technology 
Lausanne, Switzerland

AndersBjorklund,GoranHermeren,StenEirikJacobsen,
OlleLindvall,ZaalKokaia
Lund University 
Lund, Sweden

ClareBlackburnAlexanderMedvinsky,SimonTomlinson,
ValWilson,BrianHendrich
University of Edinburgh
Institute for Stem Cell Research
Edinburgh, UK

OlivierBrustleandFrankEdenhofer
University of Bonn Medical Centre
Bonn, Germany

MargaretBuckingham,AnaCumano,Jean-François
Nicolas,ShahragimTajbakhsh,BenoîtRobert
Institut Pasteur
Paris, France

ElenaCattaneoandLucianoConti
Milano University
Milan, Italy

GiulioCossu
Milano Hospital SCRI
Milan, Italy

AndersHaegerstrand
NeuroNova AB
Stockholm, Sweden

MengLi
Imperial College 
London, UK

JohnMcCafferty
Wellcome Trust Sanger Institute
Cambridge, UK

ClausNerlovandLilianaMinichiello
EMBL-Monterotondo
Rome, Italy

LarsWahlberg
NsGene A/S
Ballerup, Denmark

FionaWatt
Cancer Research UK
Cambridge, UK

ChristianDani
CNRS
Nice, France

TariqEnver
Weatherall Institute of Molecular Medicine
Oxford, UK

NewTherapy_02.indd   21

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21

gENOSTEM 

Adult mesenchymal stem cells engineering for connective tissue disorders:
fromthebenchtothebedside

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LSHB-CT-2003-3161
IntegratedProject

ECcontribution  e 872000

Startingdate 1January2004
Duration 48months

Website  www.genostem.org

Background and objectives:

Regeneration  takes  place  in  the  body  through-
out  life.  However,  bone,  cartilage,  tendons,  ves-
sels and cardiac muscle have a limited capacity 
for self-repair, and after injury or disease the re-
generative power of adult tissue is often not suf-
ficient,  leading  to  non-functional  scaring. When 
organs or tissues are irreparably damaged, they 
may be replaced by an artificial device or donor 
organ.  However,  the  number  of  available  donor 
organs is critically limited.

Generation  of  tissue-engineered  replacement 
organs  by  means  of  ex  vivo  culture  of  autolo-
gous organ-specific cells seeding a scaffold may 
represent  a  suitable  substitute  for  donor  organ 
implantation. It also prevents the risks of disease 
transmission  from  donors,  a  common  problem 
in organ implantation. Tissue engineering using 
stem cells takes full advantage of the high pro-
liferative  capacity  and  the  multi-potentiality  of 
these immature cells. The use of embryonic stem 
cells  is  limited  because  of  ethical  drawbacks,  as 
well as the allogeneic immune response, induc-
ing  rejection  of  the  implants.  The  alternative  is 
the use of adult stem cells. 

The goal of GENOSTEM is to establish a European 
international  scientific  leadership  for  stem  cell 
regenerative medicine in the field of connective 
tissue disorders. Autologous adult Mesenchymal 
Stem Cells (MSCs) are optimal candidates to serve 
as  building  blocks  for  the  engineering  of  con-

nective tissues, since they are the multipotential 
stem cells that give rise to skeletal cells (osteob-
lasts, chondrocytes and tenocytes), vascular cells 
(endothelial cells, pericytes and vascular smooth 
muscle  cells),  sarcomeric  muscle  (skeletal  and 
cardiac) and adipocytes.

Approach and methodology:

GENOSTEM  will  purify  and  optimise  cultures  of 
MSCs collected from bone marrow, skeletal mus-
cles,  fat  pads,  cord  blood  vasculature,  placental 
and adult peripheral blood of humans, as well as 
rodents. By combining different cells sources, iso-
lating different cell subsets, and providing better 
accessibility  of  growth  factors  (using  scaffolds, 
matrices,  microcarriers  coated  with  cytokines, 
etc.),  the  final  aim  is  to  obtain  cell  populations 
with  different  differentiation  and  proliferation 
potentials: 

•  undifferentiated  MSCs  where  the  full 
differentiation  potential  would  be  pre-
served; 
committed  MSCs  that  would  express, 
• 
in  addition  to  mesenchymal  factors,  at 
least one lineage-specific marker;
•  differentiated  progeny  blocked  at  a 
certain differentiation stage; 
• 

fully differentiated progeny.

The  consortium  will  also  identify  novel  factors 
essential  for  MSC  proliferation  and  differentia-
tion  using  genomic  and  proteomic  analysis,  and 
engineered  cells  in  gain  (transfer  of  genes)  and 

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REgENERATIvE MEdICINE

loss (transfer of small interfering RNAs) of function 
studies.

To  engineer  MSCs  (or  differentiated  progeny) 
with  the  appropriate  scaffold  to  secure  repair  of 
the target tissue and to define optimal conditions 
for transplantation of MSCs in normal and patho-
logical tissue, is another approach undertaken by 
GENOSTEM. Undifferentiated MSCs or MSC-differ-
entiated progeny will be assessed in normal and 
in pathological animal models, to investigate the 
reparative  potential  of  MSC  strains.  The  project 
strategy will include the use of sophisticated cul-
ture  conditions,  using  biodegradable  scaffolds, 
matrices and microcarriers binding soluble growth 
factors, inhibitors and adhesive glycoproteins and 
vectors  containing  gene  sequences  adapted  to 
the pathological state under study. Gene transfer 
will be carried out using original lentiviral vectors, 
non-viral DNA delivery complexes (polymersomes) 
or matrix-anchoring, DNA-binding peptides.

The project will define the best strategy for trans-
lating GENOSTEM research into phase I clinical tri-
als in bone defect, in partnership with SMEs and 
regulatory bodies for the scaling-up of safe proce-
dures. GENOSTEM will be taking advantage of the 
experience  already  acquired  by  one  of  the  part-
ners, in clinical trials using cultured muscle cells.

Main findings:

GENOSTEM partners have compared in vivo the 
different  scaffolds  with  MSCs,  including  hydro-
gel,  chitosan,  polymer,  PAMs  and  ceramics.  The 
project  partners  developed  procedures  that 
could  combine  cell  attachment  efficiency  with 
local  growth  factor-releasing  capacity.  Further-
more,  a  large-scale  in  vivo  model  for  cartilage 
repair  was  developed:  a  chitosan-poly(butylene 
succinate) (PBS)(50/50%wt) blend was produced 
and  processed  into  scaffolds,  using  melt  spin-
ning and fibre bonding. This scaffold was used in 
the merino sheep model for cartilage repair with 
a chondral defect. The partners have also devel-

Figure 1: Stem cells injected in merinos sheep differenti-
ating to cartilage, visualised in vivo with confocal celviso 
system (Photo Inserm)

oped scaffolds with a DNA delivery system, and a 
specific  microsphere  able  to  deliver  differentia-
tion factors. 

Standard conditions for culturing adult stem cells 
from  umbilical  cords,  adipose  tissue  and  bone 
marrow  have  been  developed.  The  cells  were 
cultured in vitro and infected with Adeno-Smad8 
and BMP-2 at different ratios, or just with Adeno-
Smad8,  and  seeded  on  a  Collagen  sponge  and 
implanted  intramuscularly  in  NOD/SCID  mice. 
Tendon-like tissue was generated with the com-
bination  of  Smad8  and  BMP-2  expression.  The 
factors involved in chondrogenesis and the engi-
neering of the cells were successfully identified. 

Chitosan  scaffolds  combined  with  TGF-treated 
ovine  stem  cells  have  been  injected  in  merino 
sheep and followed for 2 months (Fig 1). Results 
obtained with this pertinent osteoarthritis model 
have validated the protocol, and will allow clini-
cal development of the procedure. 

Moreover, the consortium has focused on bone 
repair for the setting-up of clinical trials. For this, 
animal  models  of  femoral  bone  defect  have 
been initiated. The partners developed a cranial 
critical size defect assay in nude mice for in vivo 
implantation.  A  femur  critical  size  defect  model 
using  a  novel  external  fixation  device  is  avail-

NewTherapy_02.indd   23

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New Therapies – Regenerative Medicine

23

REgENERATIvE MEdICINE

gENOSTEM 

able in immunodeficient mice for studying bone 
repair.  The  analysis  of  the  osteogenic  potential 
of  various  hMSCs  implanted  with  HA/TCP  (with 
various %) and different materials (hydrogel/ce-
ramic scaffold, chitosan) has been performed in 
this heterotopic assay in vivo. For bone induction, 
the partners identified IGF2 and IGF-BP2 through 
the microarray analyses. Treatment with IGF2 or/
and  IGF-BP2  at  the  appropriate  dosage,  slightly 
increased  ALP  staining,  suggesting  osteoblastic 
differentiation. 

Large-scale GMP production of autologous MSC 
for clinical purposes is available.

Major publications

Hoffmann,  A.,  Pelled,  G., Turgeman,  G.,  Eberle,  P., 
Zilberman, Y., Shinar, H., Keinan Adamsky, K., Win-
kel,  A.,  Shahab,  S.,  Navon,  G.,  Gross,  G.,  Gazit,  D., 
‘Neotendon formation induced by manipulation 
of  the  Smad8  signalling  pathway  in  mesenchy-
mal stem cells’, J. Clin. Invest, 2006, 116(4):940-52.

Review articles Special Focus: Mesenchymal Stem 
Cells, Regenerative Medicine, 2006, 1 (4):517-604
• 
Jorgensen  C.  Tissue  engineering 
through  mesenchymal  stem  cells:  role  of 
the Genostem Consortium
•  Delorme  B,  Chateauvieux  S,  Charbord 
P. The concept of mesenchymal stem cells.
•  Roche S, Provansal M, Tiers L, Jorgensen 
C, Lehmann S. Proteomics of primary mes-
enchymal stem cells
•  Srouji S, Kizhner T, Livne E. 3D scaffolds 
for  bone  marrow  stem  cell  support  in 
bone repair
•  Djouad F, Mrugala D, Noël D, Jorgensen 
C. Engineered mesenchymal stem cells for 
cartilage repair
•  Marie PJ, Fromigué O. Osteogenic dif-
ferentiation  of  human  marrow-derived 
mesenchymal stem cells
•  Kimelman N, Pelled G, Gazit Z, Gazit D. 
Applications  of  gene  therapy  and  adult 

stem cells in bone bioengineering
•  Hofmann A, Gross G. Tendon and liga-
ment engineering: From cell biology to in 
vivo application
•  Mendez-Peruz M, Hughes C, Annenkov 
A,  Daly  G,  Chernajovsky  Y.  Engineering 
stem cells for therapy
•  Vilquin JT, Rosset P. Mesenchymal stem 
cells in bone and cartilage repair: current 
status. 

Snedeker, J.G., Pelled, G., Zilberman, Y., Gerhard, F., 
Muller, R., Gazit, D., ‘Endoscopic cellular microsco-
py for in vivo biomechanical assessment of ten-
don function’, J Biomed Opt, 2006, 11(6):064010.

Chernayovsky,  Y.,  ‘Gene  therapy  for  arthritis 
-  where  do  we  stand?’  Arthritis  Res  Ther,  2005, 
7(6):227-9.

Arnulf, B., Lecourt, S., Soulier, J., Ternaux, B., Lacas-
sagne,  M.N.,  Crinquette,  A.,  Dessoly,  J.,  Sciaini, 
A.K., Benbunan, M., Chomienne, C., Fermand, J.P., 
Marolleau, J.P., Larghero, J., ‘Phenotypic and func-
tional  characterization  of  bone  marrow  mesen-
chymal  stem  cells  derived  from  patients  with 
multiple myeloma’, Leukemia, 2007, 21(1):158-63. 
Epub 2006 Nov 9.

Kulbe, H., Thompson, R., Wilson, J.L., Robinson, S., 
Hagemann, T., Fatah, R., Gould, D., Ayhan, A., Balk-
will, F., ‘The inflammatory cytokine tumor necro-
sis  factor-alpha  generates  an  autocrine  tumor-
promoting  network  in  epithelial  ovarian  cancer 
cells’, Cancer Res, 2007,  67(2):585-92.

Bianco,  P.,  Kuznetsov,  S.A.,  Riminucci,  M.,  Gehron 
Robey,  P., ‘Postnatal  skeletal  stem  cells’,  Methods 
Enzymol, 2007, 419:117-48.

Delorme,  B.,  Charbord,  P.,  ‘Culture  and  charac-
terisation of human bone marrow mesenchymal 
stem cells’, Methods in Molecular Medicine – Tissue 
Engineering 2nd Edition. Edited by Hansjörg Haus-
er and Martin Fussenegger. (in press)

24

New Therapies – Regenerative Medicine

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REgENERATIvE MEdICINE

Coordinator

ChristianJorgensen
INSERM
Unit 475
Hôpital Lapeyronie, Service Immuno-Rhumatologie
34000 Montpellier, France
E-mail : jorgens@montp.inserm.fr

Partners

PierreMarie
INSERM - U349 
Hôpital Lariboisiere
Paris, France

Jean-ThomasVilquin
INSERM – U582
Institut de MyologieGroupe Hospitalier Pitié-Salpêtrière
Paris, France

ClaudiaMontero-Menei
INSERM – U646
Laboratoire de Vectorisation Particulaire
Angers, France

PierreCharbord
Université François Rabelais
Laboratoire d’Hématopoïèse, Faculté de Médecine
Tours, France

YutiChernajovsky
University of London
Queen Mary and Westfield College
Bone & Joint Research Unit, Barts and The London
London, UK

AntoinetteHatzfeld
Centre National de la Recherche Scientifique (CNRS)
UPR 9045 - Laboratoire de Biologie des Cellules Souches 
Humaines
Villejuif, France

LouisCasteilla
CNRS- UMR 5018
Toulouse, France

RalphMüller
ETH - Swiss Federal Institute of Technology
Institute for Biomedical Engineering
Zürich, Switzerland

DanGazit
The Hebrew University of Jerusalem
Skeletal Biotechnology Lab
Jerusalem, Israel

GerhardGross
Helmholtz Zentrum für Infektionsforschung
Department of Gene Regulation and Differentiation
Braunschweig, Germany

PaoloBianco
Università di Roma La Sapienza
Institute for Cell Biology and Tissue Engineering
Rome, Italy

VirgilPaunescu
University of Medicine and Pharmacy Victor Babes 
Department of Physiology and Immunology 
Timisoara, Romania

RobertOostendorp
Technische Universität München
Labor für Stammzellphysiologie 
Munich, Germany

UlrikeNuber
Lund University
Stem Cell Centre
Lund, Sweden

ErellaLivne
Technion - Israel Institute of Technology
Anatomy and Cell Biology Faculty of Medicine Technion 
Haifa, Israel

NewTherapy_02.indd   25

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New Therapies – Regenerative Medicine

2

REgENERATIvE MEdICINE

gENOSTEM 

ThomasHäupl
Charité, Medical Faculty
Department of Rheumatology 
Berlin, Germany

ChristianeDascher-Nadel
Inserm-Transfert SA
Department of European and International Affaires
Marseille, France

JerónimoBlanco
Centro de Investigación Cardiovascular
Barcelona, Spain

JeffreyHubble
EPFL - Swiss Federal Institute of Technology
Institute for Biological Engineering and Biotechnology
Lausanne, Switzerland

EleniPapadaki
University of Crete
Medical School
Heraklion, Crete, Greece

MariaKalmanti
University of Crete
Heraklion, Crete, Greece

Jean-PierrePujol
Université de Caen
Laboratoire de Biochimie du Tissu Conjonctif
Caen, France

NunoNeves
University de Minho
Department of Polymer Engineering
Braga, Portugal

Jean-PierreMouscadet
Abcys SA
Paris, France

JohnWatson
Kuros Biosurgery AG
Zürich, Switzerland

NathalieRougier
Biopredic International
Rennes, France

AndresCrespo
GENOPOIETIC
Miribel, France

26

New Therapies – Regenerative Medicine

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REgENERATIvE MEdICINE

OSTEOCORd

Bone from blood: Optimised isolation, characterisation and osteogenic
inductionofmesenchymalstemcellsfromumbilicalcordblood






ContractNo 
Projecttype

LSHB-CT-200-018999
SpecificTargetedResearchProject

ECcontribution  e 200000

Startingdate 1January2006
Duration 36months

Website  www.bonefromblood.org

Background and objectives:

Stem  cells  are ‘blank’  cells  that  can  replicate  in-
definitely,  or  given  the  right  triggers,  grow  into 
specialised cells for specific areas of the body. In 
adults,  a  few  types  of  cell,  such  as  blood,  bone 
marrow or nerves, are unable to replicate them-
selves by normal cell division. Within these parts 
of the body, a small quantity of stem cells is found, 
that is used to repair and replace damaged cells. 
The  intrinsic  ability  of  stem  cells  to  self-repair 
on  demand  has  generated  a  significant  level  of 
interest in determining how scientists might be 
able to exploit stem cells for the treatment of cer-
tain disease conditions.

There  is  an  urgent  clinical  requirement  for  ap-
propriate  bone  substitutes  that  are  able  to  re-
place current grafting procedures for the repair 
of  diseased  or  damaged  bone.  Mesenchymal 
stem  cells  (MSCs)  are  found  predominantly  in 
the  bone  marrow  and  are  able  to  differentiate 
into osteogenic (bone), chondrogenic (cartilage), 
adipogenic  (fat)  and  tenogenic  (tendon)  tissue 
types,  thus  offering  considerable  therapeutic 
potential  for  tissue  engineering  applications  in 
orthopaedic surgery. 

However,  invasive  extraction  procedures  and 
poor  cell  yields  have  demanded  the  identifica-
tion of alternative tissue sources of MSCs. Grow-
ing evidence suggests that umbilical cord blood 
(UCB) contains a population of rare MSCs that are 
able  to  give  rise  to  many  different  cell  types  in 

a manner similar to bone marrow-derived MSCs.  
The aim of this project is to optimise the isolation 
and  expansion  of  MSCs  from  human  UCB  (CB-
MSCs).  The  differentiation  capacity  of  CB-MSCs 
will be examined, with a specific focus on osteo-
genesis, and we will tissue engineer 3-D bone re-
placement structures. 

Approach and methodology:

OsteoCord is a nine-partner EC FP7 project with 
representatives from academia and industry. The 
CB-MSCs  will  be  characterised  using  a  range  of 
techniques,  including  examination  of  the  gene 
and protein expression profiles, before and after 
osteogenic  differentiation,  compared  to  MSCs 
isolated  from  human  bone  marrow,  as  well  as 
embryonic  stem  cells.  The  integrated  datasets 
will allow the consortium to identify specific and/
or novel signalling pathways associated with CB-
MSCs, that will help them to understand CB-MSC 
biology  and  may  help  identify  new  targets  for 
cell-based therapies. 

Bioimpedance  measurements  of  CB-MSCs  in  2-
D  and  3-D  growth  configurations  will  be  deter-
mined, using purpose-built microchips for high-
throughput characterisation. The immune status 
of  CB-MSCs  will  also  be  determined.  Allogeneic 
transplantation of MSCs between different indi-
viduals  may  be  possible,  as  MSCs  appear  to  be 
’immune-privileged’,  in  that  they  are  not  neces-
sarily rejected when implanted into unmatched 
recipients. It is important to point out that MSCs 

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Main findings:

Since its launch, the OsteoCord project and all of 
its work packages have gained ground. The ma-
jor achievements , are set out below:

•  determined growth characteristics and 
CD antigen expression profile of CB-MSCs, 
following recovery from cryopreservation;
•  determined  electrochemical  imped-
ance measurements of BM-MSCs, using a 
planar electrode chip system;
•  developed BM-MSC spheroids for 3-D 
electrode chip assays;
•  optimisation  of  MSC  alloreactivity  as-
says in progress;
•  analysed  dynamic  microcarrier-assist-
ed bioreactors for CB-MSC growth;
•  established osteogenic differentiation 
protocols;
identified  candidate  osteogenic  scaf-
• 
folds and initiated biocompatibility studies; 
completed  an  extensive  international 
• 
patents  review,  on  the  therapeutic  appli-
cations of MSCs.

OSTEOCORd

display immuno-suppressive characteristics, and 
are  able  to  reduce  an  immune  response  and 
promote the engraftment of different cell types, 
such as skin cells and blood cells. Therefore, it is 
necessary to determine how CB-MSCs react un-
der different immune environments. 

To deliver sufficient cell numbers for viable ther-
apies, key components of the project are focused 
on  expansion  protocols.  Comparative  analyses 
of  growth  rates  and  ageing  characteristics  will 
identify the lifespan of CB-MSCs in culture. Novel 
techniques will be combined with scale-up pro-
cedures and the generation of CB-MSC lines for 
banking,  following  optimised  cryopreservation 
protocols. Biocompatibility assays using a range 
of bespoke, biomimetic scaffolds will be used to 
develop tissue engineering applications. 

An  independent  ethical  evaluation  of  the  work 
will  determine  how  the  work  has  contributed 
to the ethics of stem cell research and other is-
sues  such  as  cord  blood  banking.  OsteoCord 
will  also  provide  a  social  science  assessment  of 
the  different  expectations  of  MSCs  held  across 
the research community. The aim is to assist the 
research  community  in  better  understanding 
broader  technical,  regulatory,  commercial  and 
clinical factors in the future shaping of MSCs, and 
articulate,  robust  and  empirically  informed  sce-
narios for the exploitation of MSCs.

Expected outcome:

OsteoCord’s  integrated  approach  using  exist-
ing and complementary expertise will provide a 
timely and thorough evaluation of CB-MSCs, and 
define  appropriate  routes  for  their  therapeutic 
implementation.  The  work  ranges  from  funda-
mental biological understanding through to the 
delivery  of  CB-MSC-based  therapies,  under  ap-
propriate regulatory conditions.

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REgENERATIvE MEdICINE

HelderCruz
ECBio
Cell biotechnologies
Oeiras, Portugal

RobinQuirk
RegenTec Ltd
Nottingham, UK 

Coordinator

PaulGenever
University of York
Biomedical Tissue Research
Department of Biology (Area 9)
York, YO10 5YW, UK
E-mail: pg5@york.ac.uk 

Partners 

MoustaphaKassem
University Hospital of Odense
Department of Endocrinology
Odense, Denmark

JensAndersen
University of Southern Denmark
Department of Biochemistry and Molecular Biology
Odense, Denmark

HagenThielecke
Fraunhofer Institute for Biomedical Engineering
Biohybrid Systems Department
St Ingbert, Germany

LeeButtery
University of Nottingham
Tissue Engineering Group
School of Pharmacy
Nottingham, UK

KarenBieback
Ruprecht-Karls-Universität Heidelberg
Institute of Transfusion Medicine and Immunology
Stem Cell Laboratory
Mannheim, Germany

TrevorWalter
Angel Biotechnology Ltd
Northumberland, UK

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29

THERCORd

Development and preclinical testing of cord blood-derived cell therapy
products

REgENERATIvE MEdICINE

ContractNo 
Projecttype

LHSB-CT-2006-018817
SpecificTargetedResearchProject

ECcontribution  e 1800000
Startingdate 1May2006
Duration 36months






Website  www.thercord.eu

Background and objectives:

As  scientists  continue  to  discover  new  applica-
tions  for  human  stem  cells,  they  are  targeting 
diseases with a significant need for more effica-
cious  treatment  options.  Until  recently,  patients 
affected by acute renal failure (ARF) were treated 
by  dialytic  and  pharmacological  approaches 
therapies, with very limited efficacy. Human cord 
blood (CB) mesenchymal stem cell (MSCs) could 
represent  an  interesting  alternative  source  for 
therapeutic purposes in renal repair. 

Approach and methodology:

The  Thercord  consortium  isolated  MSCs  from 
full-term  umbilical  CB  to  test  their  therapeutic 
potential on renal tissue in vitro and in vivo. These 
cells  were  extensively  characterised  by  flow  cy-
tometry,  and  the  differentiation  assays  were 
performed towards adipogenic, osteogenic and 
chondrogenic lineages in order to confirm their 
mesenchymal  features.  Moreover,  it  tested  their 
ability to migrate in the presence of in vitro dam-
age, and to produce soluble factors to promote 
renal repair. Their ability to promote renal repair 
when  transplanted  into  NOD-SCID  mice  with 
acute renal failure was also assessed.

Main findings:

MSCs,  the  greater  the  chances  of  a  successful 
transplant and long-term engraftment. Testing of 
the  MSCs  also  confirmed  certain  characteristics 
that  help  induce  tissue  repair,  like  the  develop-
ment of bone and cartilage.

CB  MSCs  were  positive  for  CD44,  CD105,  CD90, 
HLA  class  I  and  negative  for  CD31,  CD45,  CD34, 
HLA  class  II.  Moreover,  within  the  CB  MSCs,  the 
consortium  identified  a  high  percentage  of 
CD146+/34-/45- cells, consistent with the perivas-
cular/pericyte-like  phenotype,  a  new  stem  cell 
subpopulation. In the migration assay, they were 
able to observe that these cells were capable of 
migrating and integrating into the damaged re-
nal cell line. 

With  the  proteome  assay,  they  analysed  several 
solubles,  including  Hepatocyte  Growth  Factor, 
Vascular  Endothelial  Growth  Factor,  Fibroblast 
Growth  Factor  —  factors  well  known  to  be  in-
volved  in  renal  protection  and  differentiation. 
They  tested  the  CB  MSCs  in  immunocompro-
mised mice with acute renal failure, showing that 
these cells strongly protect the mice from renal 
function  impairment.  Kidneys  were  assessed 
functionally  (blood  urea  nitrogen:  BUN),  as  well 
immunohistochemistry  and 
as  by  histology, 
Tunel  assay,  effectively  showing  lower  levels  of 
BUN, reduced tubular changes and reduced ap-
optosis in animals treated with CB MSCs. 

The  group  successfully  isolated  MSCs  from  CB 
units at a rate of approximately 18%. What is clear 
is  that  the  greater  the  percentage  of  extracted 

Interestingly, inflammatory cytokines such as IL-
alpha, IL-beta and TNF beta were statistically re-

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REgENERATIvE MEdICINE

duced in mice receiving CB MSCs, in comparison 
to  the  control  group.  In  conclusion,  they  dem-
onstrated that CB MSCs produce soluble factors 
that  play  a  critical  role,  providing  a  protective 
and  reparative  effect  in  acute  renal  failure.  And 
in  vivo  CB  MSCs  exhibit  reparative  potential  in 
acute renal failure. 

Coordinator 

LorenzaLazzari
Fondazione Ospedale Maggiore Policlinico
Mangiagalli e Regina Elena
Milan, Italy
E-mail: ctitricerche@policlinico.mi.it

Partners

MaurizioPesce
Centro Cardiologico Monzino
Milan, Italy

DominiqueBonnet
Cancer Research UK
London, UK

AnnaMagdaleneWobus
Insitut fur Pflanzengenetik und Kulturpflanzenforschung
Gatersleben, Germany

WillemFibbe
Leiden University Medical Centre
Leiden, Netherlands

JoanGarcia
Barcelona Cord Blood Bank
Barcelona, Spain

MarcelaContreras
National Blood Service
London, UK

RitaMaccario
IRCCS Policlinico San Matteo di Pavia
Pavia, Italy

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New Therapies – Regenerative Medicine

31

EPISTEM

Role of p63 and related pathways in epithelial stem cell proliferation and
differentiationandinrareEEC-relatedsyndromes

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LSHB-CT-200-019067
IntegratedProject

ECcontribution  e 8130000

Startingdate 1January2006
Duration 48months

Website  www.epistem.eu

Background and objectives:

Approach and methodology:

The  focus  of  EPISTEM  is  on  generating  new 
knowledge  and  translating  it  into  applications 
that  enhance  human  health.  To  this  end,  both 
fundamental  and  applied  research  will  be  in-
volved. EPISTEM integrates multidisciplinary and 
coordinated efforts to understand the molecular 
basis  of  factors  involved  in  epidermal  stem  cell 
generation,  maintenance  and  differentiation,  as 
well as skin disease. Moreover, the core molecule 
that will be studied in this Integrated Project (IP) 
is p63 (and related pathways), a molecule geneti-
cally proven to be involved in the development 
of rare skin diseases. 

Collectively,  the  prevalence  of  ectodermal  dys-
plasia syndromes (EDS) is estimated at 7 cases in 
10 000 births. Currently, there is no cure for these 
patients.  By  creating  the  EPISTEM  consortium, 
the  project  partners  will  address  from  different 
angles  (genetics,  gene  profiling,  molecular  and 
cellular  biology,  structural  biology,  drug  design, 
bioinformatics) the molecular pathways involved 
in epidermal dysplasia syndromes, making use of 
different technologies (mutation analysis, micro-
array,  ChiP,  transgenes,  proteomics,  in  vitro  skin 
cultures,  crystallography,  etc.).  The  consortium 
brings together leading European clinicians, ge-
neticists, molecular and cellular biologists, struc-
tural  biologists,  a  drug  designer  and  bioinfor-
matics specialists in the field of p63 (and related 
molecules) research.

EPISTEM is collecting and culturing keratinocytes 
from EDS patients with p63 mutations, as well as 
carrying out an extensive analysis of phenotype-
genotype  correlation.  These  keratinocytes  will 
be used to uncover the role of p63 proteins and 
pathways in normal and abnormal skin develop-
ment.

The consortium is building relevant in vitro and in 
vivo skin disease models for studying the role of 
p63 in EDS disease, and the genetic assessment 
of novel pathways discovered during this project. 
These models will be used for drug assessment 
as well. It will also provide insight into the regu-
lation and involvement of p63 and related path-
ways in skin differentiation, the maintenance of 
the proliferative capacity of epithelial stem cells 
and the transition of ectodermal cells to epider-
mal stem cells.

Screening  for  and  design  of  novel  therapeutic 
drugs, based on three dimensional p63 models, 
which  will  refold/reactivate  or  inhibit  p63  mu-
tants and induce biological responses in relevant 
disease models, is also key.

Expected outcome:

First  of  all,  the  EPISTEM  consortium  will  gener-
ate thorough insight into the molecular biology 
of a rare disease such as EDS, for which no cure 
is currently available. Second, characterising and 

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REgENERATIvE MEdICINE

understanding  how  epidermal  stem  cell  main-
tenance is regulated by p63 could be beneficial 
for  the  treatment  of  burn  victims,  since  these 
stem cells could be used for tissue regeneration. 
Therefore,  as  currently  estimated,  the  EPISTEM 
research proposal may have a far broader impact 
on  clinical  practice  and  for  biomedical  industry 
in the long run. Third, this integrated project will 
generate knowledge and technology that is ap-
plicable not only to p63 itself, but also to its family 
members p53 and p73; p53 being an important 
target that is mutated in most cancers and p73 
being  an  important  molecule  for  neurogenesis. 
Fourth,  from  a  technological  point  of  view,  the 
EPISTEM proposal will invest in the development 
of chIP (chromatin immunoprecipitation) on chip 
technology.

Main findings:

The  project  partners  have  collected  material 
from 226 patients in 119 families, all with muta-
tions  in  the  p63  gene.  Expression  vectors  suit-
able for transient expression in mammalian cells 
are available for 6 of the p63 isoforms (ΔN and TA 
combined  with  alpha,  beta  and  gamma). Trans-
activation assays have been carried out for sev-
eral  p63  mutations  found  in  patients.  EPISTEM 
has  generated  two  transgenic  mice  lines  over-
expressing  TAp63Δ  and  two  lines  overexpress-
ing ΔNp63Δ under the K5 promoter p63 wild-type 
background. The consortium recently developed 
a  model  of  ectodermal/epidermal  differentia-
tion from mouse embryonic stem (ES) cells, and 
showed that BMP4 leads to the apoptosis of ES-
derived  neuroectodermal  cells  while  inducting 
epidermal fate.

Target genes for specific p63 isoforms selectively 
expressed  in  proliferating  and  differentiating 
keratinocytes have been identified. EPISTEM has 
validated several p63 target genes identified by 
the gene array in vivo, using the transgenic mice 
and  the  TAp63  and/or  DNp63  genetically  com-
plemented  mice. The  partners  have  shown  that 

Figure 1: Various combinations of ectodermal dysplasia, 
orofacial clefting and limb malformations are the hallmark of 
p63-associated syndromes. EEC syndrome is the prototype 
of these syndromes and together with LMS shows all three 
hallmarks. ADULT syndrome patients never show orofacial 
clefting, whereas AEC and RHS never show limb defects. 
Non-syndromic limb defect condition (SHFM4) and non-syn-
dromic cleft lip/palate (NSCL) are also caused by mutations 
in the p63 gene.

p63  functions  as  a  negative  regulator  of  p21 
expression  in  keratinocytes  through  an  unex-
pected and complex mechanism that involves a 
reciprocal  negative  cross-talk  between  the  p63 
and Notch signaling pathways.

In order to determine the role of caspase-14, the 
consortium generated caspase-14 deficient mice. 
They determined the NMR solution structure of 
the  SAM  domain  mutant  (L518F)  which  causes 
the Hay-Wells syndrome.

Initial experiments using Saos-2 cells expressing 
exogenous temperature sensitive (ts) A167P mu-
tant TAp63Δ revealed that treatment with PRIMA-
1 inhibited cell growth in a mutant p63-depend-
ent manner, as shown by the WST-1 proliferation 
assay. Two human p63 point mutants have been 
engineered,  TAp63Δ-R204W  and  TAp63Δ-R304W. 
Both have been cloned in the pcDNAHA vector 
and in the pTRE2pur vector, to allow regulation 
by Tet (doxycyclin).

NewTherapy_02.indd   33

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New Therapies – Regenerative Medicine

33

EPISTEM

REgENERATIvE MEdICINE

Figure 2: Pathogenic p63 mutations cause at least five different syndromes and two non-syndromic conditions. Mutations 
causing different diseeases are illustrated in different colours. Only mutations that are discussed in the text are indicated, an 
overview of all mutations is given in Table 1. EEC hostpot mutations are clustered in DNA binding domain, and RHS and AEC 
syndrome mutations in SAM and TI domains. Several mutations, such as R280, R313, I510, S541 and 1850 Del A, can have a 
variable clinical outcome, probably due to genetic background effects. Tha black asterisks illustrate sites needed for upiquiti-
nation (K193, K194 and PY) and the white asterisk represents a sumoylation site (fKXD/E).40,42,58

Several data sets involving p53, p63 and p73 have 
been  collected  and  constructed.  Furthermore,  a 
web tool was developed and is now available in 
the  private  section  of  the  website  http://tools.
epistem.eu. This tool makes it possible to remain 
informed of all the datasets and the tools that are 
available.

Major publications 

Candi,  E.,  Rufini,  A.,  Terrinoni,  A.,  Dinsdale,  D., 
Ranalli,  M.,  Paradisi,  A.,  De  Laurenzi, V.,  Spagnoli, 
L.G., Catani, M.V., Ramadan, S., Knight, R.A., Melino, 
G., ‘Differential roles of p63 isoforms in epidermal 
development:  selective  genetic  complementa-
tion in p63 null mice’, Cell Death Differ, 2006, 13, 
1037-1047.

Devgan,  V.,  Nguyen,  B.C.,  Oh,  H.,  Dotto,  G.P., 
‘p21WAF1/Cip1  suppresses  keratinocyte  differ-
entiation independently of the cell cycle through 

transcriptional up-regulation of the IGF-I gene’, J 
Biol Chem, 2006, 281, 30463-30470.

Ponassi, R., Terrinoni, A., Chikh, A., Rufini, A., Lena, 
A.M., Sayan, B.S., Melino, G., Candi, E., ‘p63 and p73, 
members  of  the  p53  gene  family,  transactivate 
PKCdelta’,  Biochem  Pharmacol,  ,2006,  72,  1417-
1422.

Rinne,  T.,  Hamel,  B.,  van  Bokhoven,  H.,  Brunner, 
H.G.,  ‘Pattern  of  p63  mutations  and  their  phe-
notypes—update’,  Am  J  Med  Genet  A,  2006,  140, 
1396-1406.

Testoni, B., Borrelli, S., Tenedini, E., Alotto, D., Cast-
agnoli, C., Piccolo, S., Tagliafico, E., Ferrari, S., Viga-
no, M.A., Mantovani, R., ‘Identification of new p63 
targets in human keratinocytes’, Cell Cycle, 2006, 
5, 2805-2811.

Vigano,  M.A.,  Lamartine,  J.,  Testoni,  B.,  Merico, 

34

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REgENERATIvE MEdICINE

D.,  Alotto,  D.,  Castagnoli,  C.,  Robert,  A.,  Candi,  E., 
Melino, G., Gidrol, X., Mantovani, R., ‘New p63 tar-
gets  in  keratinocytes  identified  by  a  genome-
wide approach’, Embo J, 2006, 25, 5105-5116.

Coordinator 

PeterVandenabeele
Flanders Interuniversity Institute for Biotechnology 
VIB/UGent
Ghent, Belgium
E-mail: peter.vandenabeele@dmbr.ugent.be
or wim.declercq@dmbr.Ugent.be

Partners

HansvanBokhoven,GijsSchaftenaar,
MartijnHuynen
Radboud University Nijmegen
Nijmegen, Netherlands 

GerryMelinoandVincenzoDeLaurenzi
Università Degli Studi di Roma Tor Vergata
Rome, Italy 

GianPaoloDotto
University of Lausanne
Lausanne, Switzerland 

JohnMcGrath
King’s College London
London, UK

KlasWiman
Karolinska Institutet
Stockholm, Sweden 

VolkerDoetsch
J.W. Goethe-Universitat Frankfurt am main
Frankfurt, Germany 

RobertoMantovani
Università degli Studi di Milano
Milan, Italy 

MassimoGulisano
Istituto Oncologico del Mediterraneo
Viagrande, Catania, Italy 

NewTherapy_02.indd   35

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REgENERATIvE MEdICINE

EPISTEM

DanielAberdam
INSERM
Nice, France 

GeneSpinSrL
Milano, Italy 

PiranitKantaputra
CMU
Chiang Mai, Thailand

JingdeZhu
SJTU
Shanghai, China

36

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REgENERATIvE MEdICINE

ULCER THERAPY

Gene transfer in skin equivalents and stem cells: novel strategies for chronic
ulcerrepairandtissueregeneration

ContractNo 
Projecttype

LSHB-CT-200-12102
SpecificTargetedResearchProject

ECcontribution  e 239193
Startingdate 1July200
Duration 36months






Website  http://ulcertherapy.idi.it

Background and objectives:

Chronic skin ulceration is a frequent pathological 
condition and represents a major health problem 
for humans. In particular, cutaneous ulcers are a 
common complication of diabetes (due to mac-
rovascular  and  microvascular  disease),  and  pe-
ripheral  neuropathy,  associated  with  skin  more 
vulnerable to traumatic injuries and growth fac-
tor  expression  reduction.  Furthermore,  chronic 
ulcers  are  characterised  by  a  tissue  imbalance 
between  proteolytic  enzymes  and  their  inhibi-
tors,  often  leading  to  enhanced  growth  factor 
degradation. 

Conventional  therapeutic  approaches  (relief  of 
pressure at the wound site, surgical debridement, 
control of infection, and in selected cases, arterial 
reconstruction) are often not enough to guaran-
tee adequate healing. Non-conventional tools for 
the  treatment  of  chronic  ulcers  include  topical 
application of recombinant growth factors — di-
rectly or mediated by viral vector gene delivery 
— and transplantation of skin equivalents. How-
ever, the short half-life of exogenous growth fac-
tors, their degradation, or the inefficient delivery 
of genes to target cells, are major limitations for 
the  treatment  with  recombinant  factors.  Analo-
gously, the therapeutic effect of skin-equivalent 
grafting is limited, mostly due to infections and 
an impaired production of granulation tissue by 
the host. 

The goal of Ulcer Therapy is to assess the thera-

peutic efficacy in wound healing, of a combina-
tion of the two previous approaches, based on the 
generation and optimisation of skin equivalents, 
engineered  ex  vivo  to  over-express  therapeutic 
proteins. In parallel, the therapeutic potential in 
wound  healing  of  endothelial  progenitor  cells 
(EPCs)  and  mesenchymal  stem  cells  (MSCs),  ge-
netically modified ex vivo to express angiogenic 
molecules or genes involved in stem cell survival 
and proliferation, is under evaluation.

Approach and methodology: 

The project’s final goal will be achieved through 
the three main objectives set out below.

1.  Understanding  the  role  of  growth  factors, 
proteases, and protease inhibitors in the patho-
physiology  of  wound  healing. To  this  end,  a  ba-
sic research approach is used to characterise the 
role and mechanism of the action of potentially 
therapeutic proteins. Transgenic and conditional 
knockout  mice  are  generated  for  studying  the 
involvement  of  novel  growth  factors  in  wound 
healing.  In  parallel,  the  analysis  of  the  proteo-
lytic  degradation  of  growth  factors,  the  charac-
terisation  of  the  involved  proteolytic  enzymes, 
and the examination of presence and activity of 
tissue  protease  inhibitors  is  performed. The  cD-
NA’s  coding  for  the  studied  growth  factors  and 
protease  inhibitors  are  inserted  into  adenoviral 
vectors  that  are  used  to  deliver  these  proteins 
into wounds practised on mice. For this activity, 
animal models characterised by impaired wound 

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37

 
 
 
ULCER THERAPY

REgENERATIvE MEdICINE

Figure 1. Schematic representation of the in vivo model of 
“skin-humanised mice”. Human skin equivalents are gener-
ated from cultured primary keratinocytes and fibroblasts 
obtained from skin biopsies. Skin equivalents are then 
orthotopically transplanted onto the back of nude mice.

healing are used. Most of the study is performed 
on diabetic animals.

2.  Evaluating  the  therapeutic  potential  of  ge-
netically modified stem cells in impaired wound 
healing.  Circulating  endothelial  precursor  cells 
(EPCs)  and  mesenchymal  stem  cells  (MSCs)  se-
crete  angiogenic  growth  factors,  and  can  dif-
ferentiate  into  endothelial  cells,  thus  improving 
angiogenesis. The capacity of isolated EPCs and 
MSCs per se, or infected with adenoviral vectors 
carrying angiogenic molecules or genes involved 
in stem cell survival and proliferation, injected at 
the wound margin in mouse model of impaired 
wound  healing  (NOD/SCID  or  diabetic  mice),  is 
therefore investigated. In parallel, a broad analy-
sis is conducted to identify the most suitable ad-
enoviral  and  adeno-associated  viral  vectors  for 
gene transfer into these stem cell types.

3.  Assessing  the  safety  and  efficacy  of  geneti-
cally modified skin equivalents as a delivery sys-
tem  for  therapeutic  proteins  in  wound  healing. 
To  this  end,  quality  controls,  safety  and  efficacy 
preclinical studies in vitro and on animal models 
are performed. Fibrin is used as an optimal sub-
strate to reconstitute genetically modified com-
posite skin grafts for the proper delivery of thera-
peutic factors. After a comparative analysis of the 

Figure 2. EGFP-bioengineered “skin-humanised mice”. 
Human cultured primary keratinocytes transfected with viral 
vectors carrying EGFP are used to generate skin equivalents 
that are grafted onto nude mice. Epifluorescence 
microphotographs show the EGFP-positive epithelium, 
corresponding to the human grafted skin, surrounded by 
mouse skin.

gene transfer efficacy into keratinocytes and skin 
equivalents of adeno and adeno-associated viral 
vectors,  the  nerve  growth  factor  (NGF)  and  the 
best candidates from the genes analysed in the 
course  of  this  project,  are  employed  for  further 
in vitro and in vivo preclinical studies using skin 
grafts on immunodeficient mice.

Expected outcome:

Ulcer Therapy anticipates the following outcome: 
characterisation of the role and mech-
• 
anism  of  the  action  of  potentially  thera-
peutic proteins in wound healing;
characterisation of the proteolytic en-
• 
zyme  and  the  tissue  protease  inhibitor 
function in the ulcer environment; 
identification of the most suitable ade-
• 
noviral and adeno-associated viral vectors 
for  gene  transfer  into  keratinocytes  and 
stem cells (EPCs and MSCs);
•  assessment  of  the  quality,  safety  and 
potential  therapeutic  effects  of  geneti-
cally-modified skin equivalents;

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•  assessment  of  the  safety  and  efficacy 
in ulcer therapy of stem cells per se or in 
genetically modified form;
•  development  of  standard  operating 
procedures  for  the  ex vivo  genetic  modi-
fication of skin equivalents and their pro-
duction and testing;
•  design of a protocol for a phase I trial 
of  gene  therapy  in  humans  affected  by 
chronic diabetic ulcers.

Main findings:

The main results obtained during the first period 
of the Ulcer Therapy project are set out below:

•  The analysis of transgenic and knock-
out  mice  indicated  that  increased  ex-
pression of the angiogenic factor PlGF or 
activin or the soluble IGF-I isoform at the 
wound  site,  exerts  a  beneficial  effect  in 
wound healing. PlGF activity has also been 
investigated in diabetic mice and proved 
to  accelerate  healing  by  promoting  ang-
iogenesis.  Importantly,  adeno-mediated 
PlGF  gene  transfer  to  diabetic  wounds 
was  able  to  significantly  accelerate  heal-
ing,  approximating  the  wound  closure 
time of healthy controls. Different aspects 
of  the  repair  process  were  improved  fol-
lowing PlGF gene transfer.
•  The  analysis  of  growth  factor  degra-
dation,  protease  and  protease  inhibitors 
in the ulcer environment revealed that in 
chronic wounds PlGF and HGF, as already 
described for VEGF, are targets of proteo-
lytic cleavage that attenuates their activi-
ties.  Site-directed  mutagenesis  protected 
VEGF  from  proteolytic  degradation  and 
enhanced  its  therapeutic  activity  in  dia-
betic wound healing.
•  Adipose 
(AT-
MSCs),  which  are  easier  to  obtain  and 
propagate  in  culture  compared  to  bone 
marrow-derived  ones 
(BM-MSCs),  and 
show  capacity  to  differentiate  towards 

tissue-derived  MSCs 

different  lineages,  were  chosen  for  as-
sessing  the  efficacy  in  wound  healing  of 
MSCs.  Preliminary  data  indicated  a  role 
in promoting wound healing of AT-MSCs 
topically  administered  into  wounds  of 
diabetic mice. 
•  Of  the  different  viral  vectors  tested, 
first generation Ad5 vectors proved to be 
the most efficient in transfecting keratino-
cytes,  while  serotype-2  adeno-associated 
vectors were able to efficiently transduce 
genes into EPCs. 
•  Quality  control,  safety  and  efficacy 
studies on cultured keratinocytes and skin 
equivalents transfected with an adenovi-
ral vector containing the NGF cDNA (Ad5-
NGF-EGF)  indicated  that  high  transgene 
expression  is  achieved  without  any  rele-
vant negative effect. Moreover, the analy-
sis  of  wound  healing  in  skin  equivalents 
transduced  with  a  viral  vector  contain-
ing  the  keratinocyte  growth  factor  (KGF) 
cDNA  and  grafted  onto  nude  mice,  indi-
cated  that  KGF  overexpression  was  able 
to  enhance  closure.  This  data  indicates 
that humanised mice are a suitable model 
for testing the effect of selected factors in 
vivo.

Major publications

Cianfarani,  L.,  Zambruno,  G.,  Brogelli,  L.,  Sera,  F., 
Lacal, P.M., Pesce, M., Capogrossi, M.C., Failla, C.M., 
Napolitano, M., Odorisio, T., ‘Placenta growth fac-
tor in diabetic wound healing: altered expression 
and therapeutic potential’, Am J Pathol, 2006, 169: 
1167-1182.

Roth, D., Piekarek, M., Christ, H., Paulsson, M., Bloch, 
W.,  Krieg,  T.,  Davidson,  J.,  Eming,  S.A.,  ‘Plasmin 
modulates  VEGF-A  mediated  angiogenesis  dur-
ing wound repair’, Am J Pathol, 2006, 168:670-684.

Carretero,  M.,  Escàmez,  M.J.,  Prada,  F.,  Mirones,  I., 
Garcìa,  M.,  Holguìn,  A.,  Duarte,  B.,  Podhajcer,  O., 

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REgENERATIvE MEdICINE

ULCER THERAPY

Jorcano,  J.L.,  Larcher,  F.,  Del  Rìo,  M.,  ‘Skin  gene 
therapy for acquired and inherited disorders’, His-
tol Histopathol, 2006, 21: 1233-1247.

Eming,  S.A.,  Krieg,  T., ‘Molecular  mechanisms  of 
VEGF-A action during tissue repair’, J Investig Der-
matol Symp Proc, 2006, 11: 79-86.

Eming, S.A., Smola-Hess, S., Kurschat, P., Hirche, D., 
Krieg, T., Smola, H., ‘A novel property of povidon-
iodine: inhibition of excessive protease levels in 
chronic  non-healing  wounds’,  J  Invest  Dermatol, 
2006, 126: 2731-2733.

Perabo, L., Goldnau, D., White, K., Endell, J., Boucas, 
J.,  Humme,  S.,  Work,  L.M.,  Janicki,  H.,  Hallek,  M., 
Baker,  A.H.,  Buening,  H., ‘Heparan  sulfate  prote-
oglycan binding properties of adeno-associated 
virus retargeting mutants and consequences for 
their in vivo tropism’, J Virol, 2006, 80: 7265-7269.

Eming, S.A., Werner, S., Bugnon, P., Wickenahuser, 
C., Siewe, L., Utermöhlen, O., Davidson, J., Krieg, T., 
Roers, A., ‘Accelerated wound closure in mice de-
ficient for interleukin-10’, Am J Pathol, 2007, 170: 
188-202.

Coordinator 

GiovannaZambruno
Istituto Dermopatico dell’Immacolata, IDI-IRCCS
Lab. Biologia Molecolare e Cellulare
Via dei Monti di Creta, 104
00167 Rome, Italy
E-mail: g.zambruno@idi.it

Partners

MaurizioPesce
Centro Cardiologico Monzino
Milan, Italy

SabineWerner
Swiss Federal Institute of Technology
Zurich, Switzerland 

FernandoLarcher
Centro Investigaciones Energeticas, Medioambientales y 
Tecnologicas
Madrid, Spain 

MichaelHallek
Clinic I of Internal Medicine 
University of Cologne
Cologne, Germany

SabineEming
University of Cologne
Department of Dermatology
Cologne, Germany

NadiaRosenthal
European Molecular Biology Laboratory
Monterotondo, Rome, Italy

ElenaDellambra
IDI Farmaceutici S.p.A
Pomezia, Italy

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SKINTHERAPY

Gene therapy for edidermolysis bullosa: a model system for treatment of
inheritedskindiseases






ContractNo 
Projecttype

LHSB-CT-200-12073
SpecificTargetedResearchProject

ECcontribution  e 2079000

Startingdate 1April200
Duration 36months

Website  www.debra-international.org/researche1a.htm

Background and objectives:

Epidermolysis  Bullosa  (EB)  is  a  rare  genetic  skin 
disease, affecting approximately 30 000 individu-
als in Europe, and approximately 400 000 to 500 
000 people worldwide. The patients are predom-
inantly children, and there is no treatment avail-
able yet.

EB is characterised by an extreme fragility of the 
skin,  resulting  in  unremitting  blisters  and  ero-
sions  with  unceasing  wound  healing.  The  skin 
lesions stem from the poor adhesion of the epi-
dermis  to  the  underlying  mesenchyme,  which 
makes the skin vulnerable to damage caused by 
mild friction and trauma. The genetic bases of the 
different clinical forms of EB have been elucidat-
ed, and a precise correlation has now been made 
between genetic defects of the basement mem-
brane  components,  and  the  types  of  EB  which 
have  been  classified  within  the  Simplex  (EBS), 
Junctional (JEB), and Dystrophic (DEB) forms.

In  DEB,  generalised  blisters  heal  with  scarring. 
Extensive and progressive mutilating scarring re-
sults in increased disability and deformity of the 
fingers, as well as in contractures of the joints. The 
disease may also affect other areas of the body 
(mouth, throat, eyes), causing discomfort, and in 
most of the cases, difficulties in performing vital 
activities, like eating. 

premature  death.  In  addition  to  physical  suffer-
ing,  patients  are  faced  with  social  difficulties: 
they cannot perform physical activities, and they 
need permanent assistance and regular medical 
treatments.  EB  patients  are  also  excluded  from 
the  workforce,  not  only  owing  to  their  physical 
appearance, but also because the public is unin-
formed and unaware of the condition.

Approach and methodology: 

Skintherapy will recruit patients, and perform di-
agnoses and genetic analyses of DEB patients. A 
total of 120 patients have already been recruited. 
The  consortium  will  develop  viral  vectors  (on-
coretroviral,  lentiviral,  and  hybrid  adeno/AAV 
vectors) to integrate and firmly express the col-
lagen  VII  cDNA  in  DEB  epidermal  stem  cells  ex 
vivo; work is in progress to define the gene trans-
fer efficiency into clonogenic keratinocyte stem 
cells. Skin equivalents made with genetically cor-
rected DEB cells to assess the full morphological 
and  functional  reversion  of  the  DEB  phenotype 
will  be  constructed.  Skintherapy  will  graft  the 
skin  equivalents  expressing  the  recombinant 
collagen VII on appropriate immune competent 
animal models. The consortium is already graft-
ing and carrying out an analysis of the fate of the 
transplanted  recombinant  skin  equivalents  in 
additional dogs. 

The most severe cases are often complicated by 
the development of aggressive skin cancer and 

The  project  partners  will  develop  a  technology 
for the production and downstream processing 
of clinical-grade viral vectors preparations, under 

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SKINTHERAPY

REgENERATIvE MEdICINE

Major publications 

Mavilio, F., Pellegrini, G., Ferrari, S., Di Nunzio, F., Di 
Iorio, E., Recchia, A., Maruggi, G., Ferrari, G., Provasi, 
E., Bonini, C., Capurro, S., Conti, A., Magnoni, C., Gi-
annetti, A., De Luca, M., ‘Correction of junctional 
epidermolysis  bullosa  by  transplantation  of  ge-
netically modified epidermal stem cells’, Nat Med, 
2006, Dec;12(12):1397-402. Epub 2006 Nov 19. 

Carretero,  M.,  Escámez,  M.J.,  Prada,  F.,  Mirones,  I., 
García,  M.,  Holguín,  A.,  Duarte,  B.,  Podhajcer,  O., 
Jorcano,  J.L.,  Larcher,  F.,  Del  Río,  M.,  ‘Skin  gene 
therapy  for  acquired  and  inherited  disorders’, 
Histol Histopathol, 2006, Nov;21(11):1233-47. Re-
view.

Good  Manufacturing  and  Laboratory  Practices 
(GMP/GLP) standards. Different cell lines are be-
ing  evaluated  to  obtain  the  best  transduction 
rate and lentiviral production.

Expected outcome: 

Skintherapy’s  immediate  goal  is  to  develop  a 
gene  therapy  approach  to  DEB  and  design  ap-
propriate  phase  I/II  trials. The  expected  result  is 
a model system for DEB treatment using ex vivo 
gene  therapy.  Specifically,  the  project  aims  at 
developing  a  gene  therapy  technology  model 
based  on  autologous  transplantation  of  skin 
made in vitro, using genetically modified epider-
mal stem cells.

This  model  can  be  extended  to  other  genetic 
skin diseases requiring local or systemic delivery 
of  active  molecules. The  model  system  particu-
larly targets the development of significantly im-
proved, efficient and safe delivery systems, which 
are urgently needed.

Main findings: 

The  feasibility  of  a  phase  I/II  gene  therapy  trial 
has been evaluated on a JEB patient. Epidermal 
stem  cells  isolated  from  a  patient  affected  by 
JEB  were  transduced  using  a  retroviral  vector, 
to  make  engineered  epithelial  grafts  that  were 
transplanted  back  onto  the  patient.  Synthesis, 
proper  assembly  and  long-term  expression  of 
the  recombinant  laminin  5,  together  with  the 
development  of  a  firmly  adherent  epidermis 
with no blisters and/or infection episodes, were 
observed  for  the  duration  of  the  follow-up  (i.e. 
12 months). The results will be used to establish 
guidelines for efficient preclinical and clinical tri-
als for gene therapy of epidermal cells, and also 
for  the  development  of  a  similar  trial  for  RDEB 
and DDEB patients.

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REgENERATIvE MEdICINE

Coordinator 

GuerrinoMeneguzzi
INSERM Unité 634
27 Avenue Valombrose
06107 Nice, Cedex 02, France
E-mail: Guerrino.MENEGUZZI@unice.fr

Partners

MicheleDeLuca
Centro Regionale di Recerca Sulle
Cellule Staminali Epiteliali
Fondazione Banca Degli Occhi del Veneto
Venice, Italy

FulvioMavilio
Università di Modena e Reggio Emilia
Department of Biomedical Sciences
Modena, Italy

LeenaBruckner-Tuderman
University Hospital Freiburg
Department of Dermatology
Freiburg, Germany

MarcelaDelRio
Centro de Investigaciones Energeticas
Medioambiantales y Technologicas
Department of Epithelial Damage, Repair and Tissue 
Engineering
Madrid, Spain

AnnaStornaiuolo
Molmed S.p.A
Discovery Division
Milan, Italy

JohnDart
DEBRA Europe
Crowthorne, UK

KarineBaudin
INSERM-TRANSFERT SA
Paris, France

New Therapies – Regenerative Medicine

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THERAPEUSKIN

Ex vivogenetherapyforrecessivedystrophicepidermolysisbullosa:preclinical
andclinicalstudies

REgENERATIvE MEdICINE

ContractNo 
Projecttype

LHSB-CT-200-11974
SpecificTargetedResearchProject

ECcontribution  e 130000
Startingdate 26June200
Duration 36months

Website  www.lyon.inserm.fr/therapeuskin



/download.htm








Background and objectives:

Individuals with recessive dystrophic epidermo-
lysis  bullosa  (RDEB)  suffer  from  life-long  severe 
skin and mucosal blistering followed by scarring. 
The  disorder  causes  considerable  morbidity,  as 
well  as  premature  mortality  associated  with  an 
increased risk of squamous cell carcinomas. RDEB 
is  caused  by  loss-of-function  mutations  in  the 
type  VII  collagen  gene  (COL7A1)  encoding  the 
major structural component of anchoring fibrils 
at  the  junction  between  the  epidermis  and  the 
dermis.  No  specific  treatment  is  currently  avail-
able. The objective of the THERAPEUSKIN project 
is  to  prepare  an  ex vivo  gene  therapy  approach 
by  transplantation  of  genetically  modified  skin 
equivalents. 

Approach and methodology:

The THERAPEUSKIN project partners have devel-
oped  safe  [Self  INactivating  (SIN)]  amphotropic 
MFG-derived retroviral vectors in which COL7A1 
expression is driven by the tissue specific COL7A1 
or  the  ubiquitous  EF1alpha  promoter. The  MFG 
vector was modified due to the large size (8.9 kb) 
of the COL7A1 cDNA. Viruses were produced by 
tri-transfection  (pCOL7A1-COL7A1  and  pEF1a-
COL7A1), or by a packaging cell line (InsertAgene 
pEF1alpha-COL7A1). 

Good  transduction  efficiencies  were  obtained 
with one batch of the SINpCOL7A1-COL7A1 vec-
tor  (prepared  by  tri-transfection)  and  batches 

Figure 1. General overview of the ex vivo gene therapy ap-
proach for RDEB using SIN-COL7A1 retroviral vectors. 

of  the  SIN-pEF1alpha-COL7A1  vector  (prepared 
from  the  packaging  cell  line). The  SINpCOL7A1-
COL7A1  vector  was  subsequently  used  to  ge-
netically  correct  primary  RDEB  keratinocytes 
and fibroblasts ex vivo, which were then used to 
prepare skin equivalents (SE). The SE were made 
of a human plasma-derived fibrin gel incorporat-
ing genetically corrected RDEB fibroblasts, onto 
which genetically corrected epidermal cells were 
grown. 

Three months after grafting the genetically cor-
rected SE onto nude mice, skin biopsies showed 
a  fully  differentiated  epithelium  and  no  signs 
of blisters, as well as positive, linear type VII col-
lagen immunostaining at the dermal-epidermal 
junction,  as  well  as  ultrastructural  evidence  of 
anchoring  fibril  formation.  Assessment  of  the 

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REgENERATIvE MEdICINE

graft  after  six  months  demonstrated  the  trans-
duction of a proportion of epidermal stem cells 
and  fibroblasts  sufficient  for  the  self-renewal  of 
the  genetically  corrected  graft.  These  grafting 
experiments are currently being repeated using 
the  SIN-pEF1alpha-COL7A1  vector  produced  by 
the packaging cell line.

Expected outcome:

Reproduction  of  these  results  with  the  SIN-pE-
F1alpha-COL7A1 vector will lead to the produc-
tion of a master cell bank suitable for the prepa-
ration of clinical grade GMP vector production.

Main findings:

The  COL7A1-SIN  vectors  the  partners  have  de-
veloped have a strong therapeutic potential for 
clinical application: they have no selection mark-
ers;  they  are  self-inactivating  (reducing  the  risk 
of oncogenic events); they provide a human pro-
moter driving COL7A1 expression; and they can 
be used in an autologous SE system. A cohort of 
individuals  expressing  some  type  VII  collagen 
protein will then be selected for the grafting of 
genetically corrected skin equivalents onto lim-
ited skin areas.

Figure 2/ A-F. In vivo restoration of type VII expression and 
epidermal adherence in RDEB reconstructed skin 12 weeks 
post-grafting, using COL7A1 retroviral vectors Histological 
analysis (A,C,E) and type VII collagen immunostaining (B,D,F) 
at three months post-grafting of skin equivalents (SE) made 
of normal keratinocytes (NK) and fibroblasts (NF) (A,B), Krdeb 
and Frdeb (C,D), and genetically corrected Krdeb and Frdeb 
(E,F) with COL7A1 vector. Type VII collagen accumulates along 
the dermal-epidermal junction in normal SE and in genetically 
corrected SEs, while it is completely absent in SE made of un-
corrected RDEB cells. Note the dermo-epidermal cleft (Δ) in the 
SE made of uncorrected RDEB cells (C,D), while no blistering is 
seen in genetically corrected SE (E-F). 

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New Therapies – Regenerative Medicine

4

THERAPEUSKIN

REgENERATIvE MEdICINE

PatriciaJoseph-Mathieu
INSERM Transfert
Paris, France

Coordinator 

AlainHovnanian
INSERM U563
 Purpan Hospital
Department of Genetics
 31300 Toulouse, France
E-mail: alain.hovnanian@toulouse.inserm.fr

Partners 

MariaAntoniettaZanta-Boussif
Généthon
Evry, France

OlivierDanos
INSERM U781
Necker Hospital
Paris, France 

YannBarrandon
EPFL/CHUV
Lausanne, Switzerland

IreneLeigh
QMUL
London, UK

JohnMcGrath
KCL
St Thomas Hospital
London, UK 

ChristinaBodemer
Necker Hospital
Department of Dermatology
Paris, France

LuisMoroder
Max Planck Institute of Biochemistry
Martinsried, Germany 

JohnDart
DebRA Europe
Crowthorne, UK 

46

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REgENERATIvE MEdICINE

BETACELLTHERAPY 

BetaCellProgrammingforTreatmentofDiabetes

ContractNo 
Projecttype

LSHB-CT-200-1214
IntegratedProject

ECcontribution  e 12000000
Startingdate 1April200
Duration 60months






Website  www.betacelltherapy.org

Background and objectives:

Diabetes  is  a  prevalent  chronic  disease  that  re-
duces quality of life and increases the risks of life-
threatening  complications.  Its  onset  in  younger 
patients  is  caused  by  a  massive  loss  of  insulin-
producing  beta  cells.  Regenerating  a  functional 
beta cell mass is thus a major goal, both in bio-
medicine  and  for  society.  Beta  cell  grafts  pre-
pared from human pancreases can cure the dis-
ease, but this form of cell therapy is hindered by 
a shortage of donor organs. 

The  BetaCellTherapy  consortium,  with  leading 
teams  in  molecular,  developmental  and  func-
tional  biology,  is  conducting  an  integrated  pro-
gramme to generate insulin-producing beta cells 
in  therapeutic  quantities.  This  FP6-supported 
programme is carried out with the R&D platform 
of the JDRF (Juvenile Diabetes Research Founda-
tion) Centre for Beta Cell Therapy in Diabetes. The 
Centre organises transfer of knowledge to asso-
ciated bio-industry and multicentre clinical trials, 
as well as to the general public. It is an interna-
tional consortium composed of an R&D platform, 
a  multicentre  clinical  trial  team  and  associated 
facilities,  bio-industry  and  reference  centres;  its 
central  unit  consists  of  a  coordination  core  and 
a beta cell bank.

The  R&D  platform  explores  the  normal  biologi-
cal processes that preserve and maintain an ade-
quate beta cell mass, and applies this knowledge 
to produce functional beta cells in the laboratory 

and to regenerate them in vivo. The central unit, 
reference  centres  and  bio-industry  function  as 
translation  components  towards  clinical  imple-
mentation. A multicentre team of clinicians carry 
out  clinical  trials  on  the  prevention  and  treat-
ment  of  diabetes. Two  trials  are  currently  being 
conducted: 

•  antibody intervention in recent-onset 
type 1 diabetes; 
•  beta  cell  transplant  trial  in  advanced 
diabetes.

The Centre develops new diagnostics and thera-
peutics for these trials. It defines quality control 
and safety criteria as a guide to preclinical test-
ing.  It  also  provides  training,  and  interacts  with 
the scientific and medical community. The Cen-
tre  informs  patients  and  the  public  of  progress 
and perspectives in the field of its activities and 
objectives. It has also established a collaborative 
link to the EuroStemCell consortium, another 6th 
FP integrated project

Nature’s  biological  programme  for  develop-
ing  and  preserving  a  functional  beta  cell  mass 
throughout life, is taken as a platform for direct-
ing  strategies  towards  the  laboratory  produc-
tion of a therapeutic beta cell mass (Fig 2). Beta 
cells  will  be  derived  from  embryonic  stem  cells 
and from transdifferentiating liver, intestinal and 
pancreatic exocrine cells.

Functional  genomics  will  be  used  to  compare 
phenotypes of beta cells from new sources with 

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47

 
 
 
 
 
REgENERATIvE MEdICINE

BETACELLTHERAPY 

Embryonic Stem Cells

Endodermal Stem Cells

H epatic

Pancreatic Intestinal

P r ogenitor C ells

•  Project III: Programming a therapeutic 
beta cell mass.

These projects are based on knowledge on the 
embryonic  development  of  beta  cells;  findings 
demonstrating  plasticity  of  somatic  cells  of  en-
dodermal  origin,  suggesting  their  potential 
transdifferentiation to beta cells; and analysis of 
key  mechanisms  that  generate  and  maintain  a 
functional beta cell mass. 

Duct/A cinar C ells

Expected outcome:

Beta Cells

Functional Beta Cell Mass

Therapeutic B eta C ell Mass

for C linical T r ials of B eta C ell T her apy in Diabetes

Fig 2 - Plan of integrated programme. The numerals refer to 
the three projects.

those  isolated  from  the  pancreas.  This  analy-
sis  will  direct  further  research,  and  determine 
the start of preclinical testing. It will also gener-
ate  new  tools  and  quality  control  criteria  that 
will  allow  the  standardisation  of  ongoing  trials 
and adjustments in graft biology, to increase its 
long-term survival and function in patients. This 
project should help develop a cure for diabetes 
by (re)programming cells for beta cell therapy.

Approach and methodology:

The  overall  objectives  are  addressed  by  three 
interactive  projects  with  the  following  specific 
objectives: 

•  Project  I:  Programming  stem  cells  to-
wards beta cells;
•  Project II: Transdifferentiation of endo-
dermal cells to beta cells;

BetaCellTherapy  expects  the  following  out-
comes: 
• 
to use the physiological programming 
of  the  pancreatic  beta  cell  mass  as  tem-
plate for generating beta cells with thera-
peutic potential in the laboratory;
to collect comparative data on the effi-
• 
cacy and safety of beta cell generation by 
embryonic stem cell and transdifferentia-
tion strategies;
• 
to increase the availability of beta cell 
grafts  for  transplantation  in  diabetic  pa-
tients;
to define and disseminate quality con-
• 
trol  criteria  for  a  standardised  composi-
tion of therapeutic beta cell grafts in clini-
cal protocols;
• 
and clinical implementation;
to establish a scientific and technolog-
• 
ic platform for adjusting graft biology so 
as  to  optimise  its  long-term  survival  and 
function in patients;
• 
the consortium’s state of the art.

to transfer knowledge for bioindustrial 

to  organise  a  training  programme  on 

Main findings:

Major achievements are presented below:

ProjectI. Programming stem cells towards beta 
cells: 

48

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REgENERATIvE MEdICINE

identification of Ngn 3 targets with en-

• 
identification of steps in the transcrip-
tion factor cascade during embryonic for-
mation of pancreatic cells: HNF1ß > HNF 6 
> Pdx 1 > Nkx 6.1;
• 
docrinogenic properties: MyT1 and IA1;
• 
acid in driving endoderm to pancreas;
•  development of an in vitro model that 
recapitulates the steps of beta cell forma-
tion in rat embryonic pancreas;
•  proof-of-concept that hES cells can be 
induced into beta-like cells.

the  role  of  FGF4,  FGF10  and  retinoic 

ProjectII.Transdifferentiating endodermal cells 
towards beta cells:

transdifferentiation  of  beta-like  cells 

•  development of culture media for de-
riving  beta-like  cells  from  exocrine  pan-
creas;
• 
from human foetal liver cells;
•  discovery of insulin-expressing cells in 
the biliary duct system;
• 
transcriptome of pancreatic duct cells 
driven to neuroendocrine phenotype fol-
lowing ngn3-transfection.

Project III.  Programming  therapeutic  beta  cell 
mass:

•  assay developed to measure beta cell 
death in vitro and in vivo;
• 
transcriptome of adult rat and human 
beta  cells,  and  use  to  detect  two  novel 
beta cell, as well as specific markers, and to 
identify  beta-cell  specific  genes  in  ngn3-
transdifferentiated human duct cells;
• 
tional level;
•  development  and  phenotyping  mice 
with Irs2-deletion in pancreatic endocrine 
or beta cells;
• 
med cells for susceptibility to cytokines.

regulation  of  phenotype  at  transla-

In  vitro  model  for  screening  neofor-

Major publications:

Duvillie,  B.,  Attali,  M.,  Bounacer,  A.,  Ravassard,  P., 
Basmaciogullari,  A.,  Scharfmann,  R., ‘The  Mesen-
chyme  Controls  the  Timing  of  Pancreatic  Beta-
Cell Differentiation’, Diabetes, 2006, 55(3):582-9.

Pedersen,  J.K.,  Nelson,  S.B.,  Jørgensen,  M.C., 
Henseleit,  K.D.,  Fujitani, Y.,  Wright,  C.V.E.,  Sander, 
M.,  Serup,  P.,  ‘Endodermal  expression  of  Nkx6 
genes  depends  differentially  on  Pdx1’,  Develop-
mental Biology, 2005, 288(2):487-501

Brolen, G.K., Heins, N., Edsbagge, J., Semb, H., ‘Sig-
nals from the embryonic mouse pancreas induce 
differentiation  of  human  embryonic  stem  cells 
into insulin-producing beta-cell-like cells’, Diabe-
tes, 2005, 54(10):2867-74.

Poll, A.V., Pierreux, C.E., Lokmane, L., Haumaitre, C., 
Achouri, Y., Jacquemin, P., Rousseau, G.G., Cereghi-
ni, S., Lemaigre, F.P., ‘A vHNF1/TCF2-HNF6 cascade 
regulates  the  transcription  factor  network  that 
controls generation of pancreatic precursor cells’, 
Diabetes, 2006, 55(1):61-9.

Baeyens, L., De Breuck, S., Lardon, J., Mfopou, J.K., 
Rooman, I., Bouwens, L., ‘In vitro generation of in-
sulin-producing  beta  cells  from  adult  exocrine 
pancreatic cells’, Diabetologia, 2005, 48(1): 49-57.

Mellitzer, G., Bonné, S., Luco, R.F., Van De Casteele, 
M.,  Lenne,  N.,  Collombat,  P.,  Mansouri,  A.,  Lee,  J., 
Lan, M., Pipeleers, D., Nielsen, F.C., Ferrer, J., Grad-
wohl,  G.,  Heimberg,  H., ‘IA1  is  Ngn3-dependent 
and essential for differentiation of the endocrine 
pancreas’, Embo J, 2006; 25(6):1344-52. 

Zalzman,  M.,  Anker-Kitai,  L.,  Efrat,  S., ‘Differentia-
tion  of  human  liver-derived  insulin-producing 
cells towards the beta-cell phenotype’, Diabetes, 
2005, 54(9):2568-75.

Bogdani,  M.,  Suenens,  K.,  Bock,  T.,  Pipeleers-
Marichal, M., In’t Veld, P., Pipeleers, D., ‘Growth and 

NewTherapy_02.indd   49

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New Therapies – Regenerative Medicine

49

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
REgENERATIvE MEdICINE

BETACELLTHERAPY 

Functional Maturation of {beta}-Cells in Implants 
of Endocrine Cells Purified From Prenatal Porcine 
Pancreas’, Diabetes, 2005, 54(12):3387-94.

Casellas, A., Salavert, A., Agudo, J, Ayuso, E., Jimen-
ez, V., Moya, M., Munoz, S., Franckhauser, S., Bosch, 
F., ‘Expression of IGF-I in pancreatic islets prevents 
lymphocytic infiltration and protects mice from 
type 1 diabetes’, Diabetes, 2006, 55(12):3246-55.

Jackerott,  M.,  Moldrup,  A.,  Thams,  P.,  Galsgaard, 
E.D., Knudsen, J., Lee, Y.C., Nielsen, J.H., ‘STAT5 ac-
tivity  in  pancreatic  beta-cells  influences  the  se-
verity of diabetes in animal models of type 1 and 
2 diabetes’, Diabetes, 2006, 55(10):2705-12. 

Coordinator

DanielPipeleers
Vrije Universiteit Brussel
Diabetes Research Centre
Laarbeeklaan 103
1090 Brussels, Belgium
E-mail: daniel.pipeleers@vub.ac.be

Partners

DanielPipeleers,HarryHeimberg,LucBouwens
Vrije Universiteit Brussel
Brussels, Belgium

FrédéricLemaigre
Christian de Duve Institute of Cellular Pathology
Brussels, Belgium

BernardPeers
Université de Liège
Liege, Belgium

JensH.Nielsen
University of Copenhagen
Copenhagen, Denmark

ShimonEfrat
Tel Aviv University
Tel Aviv, Israel

FatimaBosch
Universitat Autònoma de Barcelona
Barcelona, Spain

HenrikSemb
Lund University
Lund, Sweden

PedroHerrera
University of Geneva
Geneva, Switzerland

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REgENERATIvE MEdICINE

JonathanSlack
University of Bath
Bath, UK

PetterBjörquist
Cellartis AB
Göteborg, Sweden

DominicWithers
University College London
London, UK

FinnC.Nielsen
University of Copenhagen Hospital Rigshospitalet
Copenhagen, Denmark

ZhidongLing
Academic Hospital VUB
Brussels, Belgium

YuvalDor
The Hebrew University
Hadassah Medical School
Jerusalem, Israel

ChristelHendrieckx
JDRF Centre for Beta Cell Therapy in Diabetes
Brussels, Belgium

PhilippeRavassard
Centre National de la Recherche Scientifique
Paris, France

RaphaelScharfmannandGérardGradwohl
INSERM
Paris, France

JorgeFerrrer
Institut d’Investigacions Biomediques A. Pi I Sunyer
Barcelona, Spain

AnneGrapin-Botton
Swiss Institute for Experimental Cancer Research
Lausanne, Switzerland

SueSwift
Beta-Cell NV
Brussels, Belgium

LucSchoonjans
Thromb-X NV
Louvain, Belgium

OleMadsenandPalleSerup
Novo Nordisk A/S
Gentofte, Denmark

ThomasMandrup-Poulsen
Steno Diabetes Centre
Gentofte, Denmark

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1

EUROSTEC

EuropeanprogrammeonSoftTissueengineeringforChildren

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LSHC-CT-2006-037409
IntegratedProject

ECcontribution  e 782800

Startingdate 1January2007
Duration 60months

Website  www.eurostec.eu

Background and objectives:

The aim of the EuroSTEC project is to use modern 
tissue engineering approaches to treat children 
with  structural  disorders  present  at  birth,  such 
as  spina  bifida,  urogenital  defects,  gastroschisis, 
diaphragmatic  hernia  and  esophageal  atresia. 
The  project  strives  to  take  a  translational  route 
through in vitro and animal experiments to early 
clinical  trials.  Tailor-made  ‘smart’  biomatrices 
(scaffolds)  will  be  prepared,  using  natural  scaf-
fold  molecules  (collagen,  elastin)  and/or  man-
made polymers (polylactic/glycolic acid), and will 
be substituted with regulatory molecules such as 
growth factors and glycosaminoglycans. 

A variety of cells, including stem cells, fibroblasts, 
muscle cells and urothelial/epithelial cells will be 
cultured in vitro and seeded into biomatrices. For 
major congenital birth defects, biomatrices, with 
or without cells, will be prepared and implanted 
using  novel  animal  models,  and  evaluated  for 
their  capacity  to  regenerate  the  correct  tissues. 
Biomatrices will degrade in time, and then be re-
placed by the bodies’ own tissues, thus assuring 
compliance with growth which is especially im-
portant in young children. Prenatal and postnatal 
reconstructive procedures will improve the final 
outcome of reconstructive surgery. Clinical trials 
for  diaphragmatic  hernias  will  form  the  start  of 
the  patient  registry  and  protocol  development 
for future clinical studies. 

Ethical  and  regulatory  issues  will  be  fully  ad-
dressed before final clinical application, and par-
ents and children will have to be able to under-
stand  these  new  treatment  options.  A  dialogue 
with the general public, including patient’s asso-
ciations, will be sought. Demonstration activities 
will be carried out to increase the awareness of 
new treatment modalities based on tissue engi-
neering.  Finally,  surgeons  will  be  trained  to  use 
the new operation techniques. 
The  project  combines  European  leaders  in  the 
field  of  biomatrices,  cell  culture,  animal  models, 
surgery, and ethical and regulatory issues.

Approach and methodology:

The  EuroSTEC  project  has  5  research  areas  that 
cover  different  aspects  of  soft  tissue  engineer-
ing  for  congenital  birth  defects  in  children. The 
first research area focuses on ‘biomatrices for tis-
sue engineering’. Each tissue has its own unique 
set  and  content  of  scaffold  biomolecules.  By 
selectively 
incorporating  biologically  active 
molecules,  such  as  morphogens  into  tissue-en-
gineering constructs, cellular behaviour may be 
fine-tuned. The synthesis of biodegradable sup-
port polymers can be achieved using approved 
monomers,  following  established  procedures  in 
the  laboratory.  In  this  project,  the  aim  is  to  de-
velop polymers that can carry a mechanical load 
for  periods  spanning  3  to  6  months,  and  that 
are processed to form suturable compliant sup-
ports for the above bioactive extracellular matrix 
(ECM).

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REgENERATIvE MEdICINE

In  research  area  2, ‘cell  culture  systems  and  bi-
omatrix interactions’, in vitro cell culture systems, 
cell lines and human cell culture systems are ap-
plied to study epithelial and skin tissues, muscle 
tissues, urogenital, esophagus and amniotic cells 
and  tissues.  Interactions  between  vascular  sup-
ply  and  biomatrices  have  been  investigated  for 
extracellular matrices.
Now, various cell lines with different characteris-
tics are available for study. This allows compara-
tive  analyses  of  scaffolds  of  different  composi-
tion to construct cell-matrix implants.

Several  of  the  university  research  centres  have 
developed  different  animal  models  for  severe 
congenital  structural  anomalies  which  can  be 
used for research area 3, ‘models for severe con-
genital anomalies’. These models can be used to 
study  new  treatment  strategies,  including  tis-
sue  engineering  techniques.  Currently,  models 
for  foetal  or  postnatal  investigations  are  avail-
able  for  spina  bifida  (sheep),  skin  and  epithelial 
investigations  (rat),  urogenital  reconstructions 
(rabbit, sheep), Gastroschisis and Congenital dia-

Figure 1: Flowchart showing the interactions of the dif-
ferent research areas (RAs) and management overview of 
the EuroSTEC project

phragmatic Hernia (sheep, rabbit) and oesopha-
gus atresia (sheep). In foetal models and clinical 
invasive  procedures  the  disruption  of  amniotic 
membranes  plays  an  important  role  in  second-
ary morbidity. 

Currently, children with severe structural anoma-
lies such as spina bifida, bladder extrophy, gastro-
schisis,  congenital  diaphragmatic  hernia  (CDH), 
and oesophageal atresia need extensive surgical 
procedures  with  long-term  additional  morbid-
ity. Most reconstructions consist of local skin flap 
coverage of the defects, local tissue growth of the 
organ after closure of the defects, or excision of 
dysplastic tissues and additional reconstructions. 
Scar tissue, dysplastic and ischemia can prevent 
a  good  functional  outcome  and  may  result  in 
long-term  complications.  In  EuroSTEC,  ‘Clinical 
treatments,  pilot  studies  and  instruments’  are 
part of research area 4, which includes a study of 
intervention for CDH.

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New Therapies – Regenerative Medicine

3

 
EUROSTEC

REgENERATIvE MEdICINE

In the last few decades, many European countries 
have  had  an  ongoing  debate  about  the  follow-
ing  question:  to  what  extent  is  it  desirable  and 
morally  acceptable  to  treat  these  children  with 
all kinds of technological means? This issue will 
be further studied in research area 5, ‘ethical and 
clinical registry and study management.’

Expected outcome:

EuroSTEC forms a strategic combination of basic 
molecular, cellular and tissue structure scientific 
knowledge  with  clinical  paediatric  patient  care 
and surgical experience, as well as the develop-
ment  of  new  treatment  strategies.  The  project 
combines scientific information and cooperation 
between  different  individual  scientific  groups 
working in the field of tissue engineering in Eu-
rope. Technically,  this  will  lead  to  new  products 
which  can  be  evaluated  in  a  preclinical  setting 
for applications in children with severe congeni-

Figure 2: Graphical presentation of the different research 
areas and the partners involved in the work.

tal structural anomalies needing life-long medi-
cal  care. The  involvement  of  industrial  partners 
will  improve  the  technical  aspects,  offer  larger 
production facilities and a distribution network, 
as well as quality control systems. The new ‘smart’ 
biomaterials  will  be  distributed  by  European 
companies  in  the  field  of  tissue  engineering, 
with  worldwide  applications  both  for  children 
with  different  congenital  structural  anomalies 
and  also  for  markets  in  the  field  of  biomedical 
devices involving all children’s hospitals.

The project should lead to a database with dis-
ease-specific  information  concerning  children 
with severe structural anomalies that can be cor-
related with environmental epidemiological and 
genomic  databases  for  further  elucidation  of 
underling  disease  courses. The  development  of 
this specific patient registration and study proto-

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REgENERATIvE MEdICINE

cols will enable the start of phase II studies (and 
phase II/III studies). 

EuroSTEC  will  also  lead  to  socio-ethical  discus-
sions  and  protocol  development  concerning 
the use of new biomaterials in children and the 
ethical aspects involved. The inventory of the dif-
ferent ethical aspects and diverse social implica-
tions  in  various  European  countries,  in  addition 
to the evaluation and analysis of this information, 
will  lead  to  applicable  protocols  in  all  involved 
clinical  settings,  and  solutions  for  regulatory 
problems before the start of clinical trials.

Coordinator

WouterFeitz
Radboud University Nijmegen Medical Centre
Department of Urology (659)
PO Box 9101
6500 HB Nijmegen, Netherlands
E-mail: m.bussemakers@uro.umcn.nl

Partners 

JönsHilborn
Uppsala University
Polymer Chemistry, 
Dept. of Materials Chemistry
Uppsala, Sweden

PeterFrey
Centre Hospitalier Universitaire Vaudois
Dept. of Paediatric Urology and Surgery
Lausanne, Switzerland

JanDeprest
Katholieke Universiteit Leuven
Department of Gynaecology and Obstetrics
Leuven, Belgium

GerardBarki
Karl Storz GmbH & Co.KG
Tuttlingen, Germany

MartinMeuli
University Children’s Hospital Zurich
Department of Surgery
Zurich, Switzerland

RolandZimmermann
University Hospital Zurich
Department of Obstetrics 
Zurich, Switzerland 

AmulyaSaxena
Medizinische Universität Graz
Department of Paediatric Surgery
Graz, Austria

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

REgENERATIvE MEdICINE

EUROSTEC 

WimWitjes
CuraTrial SMO & Research BV
Arnhem, Netherlands

NoesdeVries
European Medical Contract Manufacturing (E.M.C.M.) B.V.
Nijmegen, Netherlands

BenjaminHerbage
Symatese Biomateriaux
Chaponost, France

PaulvandenBerg
University Medical Centre Groningen
Department of Obstetrics and Gynaecology
Groningen, Netherlands

EduardGratacos
Institute d’Investigacions Biomediques August Pi I 
Sunyer
Foetal Medicine + Therapy Research Group
Barcelona, Spain

KyprosNicolaides
Foetal Medicine Foundation / King’s College Hospital
Harris Birthright, Foetal Medicine Unit
London, UK

IngoHeschel
Matricel GmbH
Herzogenrath,  Germany

6

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REgENERATIvE MEdICINE

SC&CR

Application and process optimisation of human stem cells for myocardium
repair






ContractNo 
Projecttype

LHSB-CT-2004-02988
SpecificTargetedResearchProject

ECcontribution  e 194200

Startingdate 1February2004
Duration 48months

Website  www.SC-CR.eu

Background and objectives:

Sudden untreated occlusion of a coronary artery, 
following plaque rupture and/or vasospasm, may 
result  in  extensive  necrosis  of  the  myocardium, 
i.e.  a  transmural  myocardial  infarction.  Because 
remaining cardiomyocytes are not able to repli-
cate, the loss of cardiomyocytes after infarction is 
irreversible, and the tissue defect heals by fibrotic 
scarring after eight weeks. Moreover, post-infarc-
tion geometric disarrangement of the myocytes 
and  extracellular  matrix,  dilation  of  ventricular 
cavity and compensatory hypertrophy of the re-
maining myocardium, a process defined through 
ventricular remodelling, may contribute to devel-
opment of ventricular dysfunction. 
Autologous  transplantation  of  bone  marrow 
stem cells into the infarcted myocardium is an in-
novative and promising strategy for the therapy 
of heart failure due to ischemic heart disease. The 
SC&CR project proposes a strongly integrated ap-
proach to verify human stem cells’ safety and ef-
ficacy for the treatment of ischemic myocardium, 
and to identify interventions that can be rapidly 
translated to clinical practice, in order to prevent 
or reduce damage to ischemic tissue. 

peutic  potential  of  these  cells.  In  general,  there 
are  several  prerequisites  to  the  validation  of 
large-scale  use  of  stem  cells  for  therapeutics, 
namely:
• 
identifying the most suitable stem cell 
type(s) to be used, to repair/regenerate a 
given organ;
• 
knowing  the  combination  of  stimuli 
that  might  drive  differentiation  of  stem 
cells toward a specific lineage;
identifying  genes  important  for  stem 
• 
cell conversion, as well as setting up safe 
and  effective  vectors  and  protocols  to 
induce  cardiac  myocyte  differentiation 
pathway(s) by gene transfer;
•  assessing the functional properties of 
stem cell-derived differentiated cells;
•  unravelling  the  action  of  factors  that 
might  be  involved  in  recruitment  and 
homing  of  endogenous  stem  cell  in  tis-
sues where regeneration is needed;
•  evaluating  the  mid-  and  long-term 
effects  of  stem  cell  administration  in  pa-
tients, after having performed exhaustive 
preliminary  analysis  of  stem  cell  use  in 
animal preclinical models. 

Approach and methodology:

Expected outcome:

Despite  the  number  of  observations  report-
ing “transdifferentiation”  of  tissue-derived  stem 
cells,  there  is  no  conclusive  evidence  on  the 
mechanism(s)  underlying  changes  in  stem  cell 
fate,  and  insufficient  information  on  the  thera-

It is expected that autologous HSC transplanta-
tion  into  the  infarcted  or  chronically  ischemic 
myocardium  may  be  highly  effective  in  wound 
repair, in terms of heart muscle regeneration and 
improvement  of  coronary  blood  flow.  The  con-

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New Therapies – Regenerative Medicine

7

 
 
 
 
 
 
 
 
 
REgENERATIvE MEdICINE

SC&CR

sortium notes that while the approach described 
in  SC&CR  —  i.e.  pharmacological  mobilisation, 
collection by means of apheresis and intra-myo-
cardial  or  intra-coronary  injection  of  stem  cells 
under direct vision — has not been used by oth-
er groups, it offers several advantages compared 
to other methods such as the collection of stem 
cells from the bone marrow by direct aspiration, 
and from the skeletal muscle by biopsy. 

Firstly,  it  is  painless,  it  does  not  require  general 
anaesthesia and it does not imply any blood loss. 
This is a major advantage because it reduces the 
overall  procedural  risk  in  certain  subsets  of  pa-
tients, like those affected by heart failure and re-
fractory angina pectoris. Thus far, no major com-
plication has emerged following the clinical use 
of  the  granulocyte  colony  stimulating  factor,  in 
patients affected by ischemic heart disease. 

There is a great deal of clinical experience of HSC 
mobilisation  and  collection  by  apheresis  in  the 
haematological  field. This  technique  is  adopted 
even in healthy donors without significant com-
plications. Additionally, HSCs do not require any 
ex vivo pharmacological manipulation or culture 
in order to modulate their differentiation poten-
tial.  Moreover,  the  number  of  collected  cells  is 
significantly higher than after bone marrow aspi-
ration (60-80 x106 vs. 10-30 x106 BMSC). 

The  intra-myocardial  injection  under  direct  vi-
sion, during a CABG procedure, allows precise an-
atomic identification of the target area, and even 
of the distribution of the injections. By contrast, 
the intravenous route is limited by the first-pass 
attenuation effect on the BMSC into the pulmo-
nary  circulation,  and  by  the  fact  that  the  myo-
cardium receives only 3-5% of the whole cardiac 
output. Consequently, the number of stem cells 
populating an infarcted or ischemic area may be 
significantly reduced. 

Finally,  the  use  of  autologous  adult  BMSC  does 
not imply any ethical issues, in contrast with em-

bryonic stem cells. The clinical use of HSCs in the 
treatment of severe forms of ischemic heart dis-
ease  may  represent  an  additional  or  alternative 
therapeutic option that may beneficially modify 
the  unfavourable  course  of  the  disease.  HSCs 
transplantation  may  be  combined  with  con-
ventional therapies in order to add further ben-
efit. Repopulating areas of muscle loss, restoring 
¬— at least partly ¬— the systolic and diastolic 
properties  of  the  left  ventricle,  and  stimulating 
the growth of new blood vessels may reduce the 
high  incidence  of  mortality  and  complications 
associated with heart disease. Both a significant 
improvement in the quality of life of the patients, 
and a decrease in the social-economic burden of 
ischemic heart disease are expected.

Main findings:

During the first reporting period of this project, 
work  was  carried  out  in  order  to  understand 
molecular  events  associated  with  cardiac  myo-
cyte  differentiation  of  stem  cells  using  cell  cul-
ture, physiology, genetic transfer, preclinical and 
clinical  approaches.  This  work  has  involved  the 
development  of  individual  activities  sustained 
by a high intra-network dissemination of results, 
and the sharing of tools and common platforms. 
Work has been carried out in four areas:

Clinical Activities: In this part of the project, two 
independent  phase  I  trials  of  autologous  bone 
marrow-derived stem cells, in patients suffering 
chronic ischemic heart disease are being carried 
out.

Physiologic  assessment  of  stem  cell  differentia-
tion: Differentiation of stem cells in the myocar-
dium  was  assessed  morphologically  in  several 
studies in different preclinical models. However, 
the extent of functional integration into the my-
ocardium  is  still  a  matter  of  debate.  In  this  part 
of the project, the consortium targeted clarifying 
at a functional level the differentiation of several 
stem cell types, using cell culture techniques. 

8

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REgENERATIvE MEdICINE

Brugh, S.A., Zahanich, I., Rüschenschmidt, C., Bekc, 
H., Blyszczuk, P., Czyz, J., Heubach, J.F., Ravens, U., 
Horstmann,  O.,  St-Onge,  L.,  Braun,  T.,  Brustle,  O., 
Boheler,  K.R.,  Wobus,  A.M., ‘Signals  from  embry-
onic fibroblasts induce adult intestinal epithelial 
cells to form nestin-positive cells with prolifera-
tion  and  multineage  differentiation  capacity  in 
vitro’, Stem Cells, 24:2085-2097. 

De  Boer,  T.P.,  Van  der  Heyden,  M.A.G.,  Rook, 
M.B.,  Wilders,  R.,  Broekstra,  R.,  Kok,  B.,  Vos,  M.A., 
De  Bakker,  J.M.T.,  Van  Veen,  T.A.B.,  ‘Pro-arrhyth-
mogenic potential of immature cardiomyocytes 
is triggered by low coupling and cluster size’, Car-
diovasc Res, 2006, 71:704-714. 

De  Boer,  T.P.,  Van  Veen,  T.A.B.,  Bierhuizen,  M.F.A., 
Kok,  B.,  Rook,  M.B.,  Boonen,  K.J.M., Vos,  M.A.,  Do-
evendans, P.A., De Bakker, J.M.T., Van der Heyden, 
M.A.G., ‘Connexin 43 repression following epithe-
lium-to-mesenchyme  transition  in  embryonal 
carcinoma cells requires Snail 1 transcription fac-
tor’, Differentiation, 2007, Mar;75(3):208-18. 

In vivo preclinical studies: The consortium’s goal 
was to assess the effect of factors and genes po-
tentially involved in the recruitment, homing, dif-
ferentiation and proliferation of stem cells in the 
infarcted heart. 

In  vitro  studies:  A  number  of  groups  belonging 
to the SC&CR consortium sought to analyse the 
differentiation of stem cells by cell culture, gene 
manipulation, genomic analysis and biopolymers 
embedding. 

Major publications

Minetti,  G.C.,  Colussi,  C.,  Adami,  R.,  Serra,  C., 
Mozzetta,  C.,  Parente,  V.,  Fortuni,  S.,  Straino,  S., 
Sampaolesi,  M.,  Di  Padova,  M.,  Illi,  B.,  Gallinari,  P., 
Steinkuhler, C., Capogrossi, M.C., Sartorelli, V., Bot-
tinelli,  R.,  Gaetano,  C.,  Pure,  P.L., ‘Functional  and 
morphological  recovery  of  dystrophic  muscles 
in  mice  treated  with  deacetylase  inhibitors’,  Nat 
Med, 2006, 12:1147-1150. 

D’Arcangelo,  D.,  Ambrosino,  V.,  Giannuzzo,  M., 
Gaetano, C., Capogrossi, M.C., ‘Axl receptor activa-
tion mediates laminar shear stress anti-apoptotic 
effects  in  human  endothelial  cells’,  Cardiovasc 
Res, 2006, 71:754-63. 

Lagostena,  L.,  Avitabile,  D.,  De  Falco,  E.,  Orlandi, 
A., Grassi, F., Iachininoto, M.G., Ragone, G., Fucile, 
S.,  Pompilio,  G.,  Eusebi,  F.,  ‘Electrophysiological 
properties of mouse bone marrow c-kit cells co-
cultured onto neonatal cardiac myocytes’, Cardio-
vascr Res, 2005, 66:482-492. 

Nikolova,  T.,  Wu,  M.,  Brumbarov,  L.,  Alt,  R.,  Opitz, 
H.,  Boheler,  Cross  M.,  Wobus,  A.M.,  ‘WNT-condi-
tioned  media  differentially  affect  the  prolifera-
tion  and  differentiation  of  cord  blood-derived 
CD133+cells  in  vitro’,  Differentiation,  2007,  74 
Feb;75(2):100-11.

Wiese,  C.,  Rolletscheck,  A.,  Kania,  G.,  Navarrete-
Santos, A., Anisimov, S.V., Steinfarz, B., Tarasov, K.V., 

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New Therapies – Regenerative Medicine

9

REgENERATIvE MEdICINE

MichaelHallek
University of Cologne Clinical Department
Cologne, Germany 

SC&CR

Coordinator 

MaurizioC.Capogrossi
Istituto Dermopatico dell’Immacolata – IDI – IRCCS
Laboratorio di Patologia Vascolare
Via dei Monti di Creta
104 – 00167 Rome, Italy
E-mail: capogrossi@idi.it 

Partners

MaurizioPesce
Centro Cardiologico Monzino
Milan, Italy

AntonioZaza
Università degli Studi Milano-Bicocca
Milan, Italy

AldonaDembinska-Kiec
The Jagiellonian University
Krakow, Poland

AnnaMagdalenaWobus
Institute of Plant Genetics and Crop Plant Research
Gatersleben, Germany

CarstenWerner
Institut für Polymerforschung Dresden e.V.
Dresden, Germany 

JacquesDeBakker
Interuniversity Cardiology Institute of the Netherlands
Utrecht, Netherlands

NadiaRosenthal
European Molecular Biology Laboratory
Monterotondo, Rome, Italy

MariaLuisaNolli
Areta International
Gerenzano, Varese, Italy 

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STEMSTROKE

Towardsastemcelltherapyforstroke






ContractNo 
Projecttype

LHSB-CT-2006-03726
STREP

ECcontribution  e 24708

Startingdate 1January2007
Duration 36months

Website  www.stemstroke.eu

Background and objectives:

Stroke  is  a  major  cause  of  long-term  disability 
in humans, and there is a lack of effective treat-
ments.  Stroke  affects  about  3.5  million  people 
in  the  EU,  with  700  000  new  cases  every  year. 
Although  most  stroke  survivors  spontaneously 
recover to some degree, more than half of stroke 
patients  suffer  significant  residual  impairments, 
creating  enormous  economic  and  societal  bur-
dens. For example, Sweden reports an incidence 
of 213 first-ever strokes per 100 000 individuals, 
indicating  that  the  total  excess  direct  and  indi-
rect cost of stroke would be Δ1.5 billion, with al-
most 45% of the direct costs attributed to social 
services. 

The extension of the average human lifespan has 
resulted in a steady increase in the incidence of 
stroke among the expanding elderly population, 
creating  a  serious  hazard  to  quality  of  life  and 
also increasing health care costs. From this per-
spective, all efforts to bring the latest advances of 
science and technology a step closer to the de-
velopment of therapies against stroke, is of great 
importance. 

The  possibility  to  isolate  and  propagate  neural 
stem cells (NSCs) with self-renewal and multipo-
tential properties from the foetal and adult nerv-
ous system, as well as embryonic stem (ES) cells 
and  their  potential  applications  in  cell  therapy, 
have attracted a lot of research interest in recent 
years. This field is just starting to reveal its poten-

tial, and many aspects related to basic biological 
properties of NSCs, and their possible application 
as  therapeutic  tools,  still  has  to  be  established. 
The limited capacity of NSCs to be expanded in 
vitro, and the enormous need to develop repro-
ducible technology to obtain a stable and suffi-
cient number of NSCs as a source for cell therapy 
in  neurodegenerative  diseases,  makes  it  impor-
tant to characterise NSCs obtained from different 
sources, and to explore their capacity to survive 
and integrate into the damaged brain.

A  novel  mechanism  for  neuronal  replacement 
after  stroke,  i.e.,  the  formation  of  new  neurons 
from  the  adult  brain’s  own  NSCs,  was  demon-
strated  recently.  New  neurons  are  generated 
in  the  subventricular  zone  and  migrate  to  the 
striatum,  where  they  differentiate  into  mature 
neurons with the characteristics of those which 
have  died. This  potential  self-repair  mechanism 
has raised a lot of interest in both the scientific 
and clinical communities. Moreover, recent data 
indicate  that  stroke-induced  neurogenesis  is 
long-lasting  and  that  endogenous  NSCs  in  the 
adult  brain  produce  new  mature  neurons  over 
several months following a stroke. The long-last-
ing neurogenesis occurs concomitantly with the 
spontaneous recovery of motor function, which 
is observed over several months in both animals 
and humans affected with stroke. Whether there 
is any causal relationship between striatal neuro-
genesis and behavioural recovery after stroke is 
as yet unknown.

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REgENERATIvE MEdICINE

STEMSTROKE

cognitive  deficits.  Finally,  the  transplantation- 
and endogenous neurogenesis-based strategies 
will be optimised in animal models of stroke, and 
with close collaboration between basic scientists 
and clinicians, a preclinical protocol for stem cell 
application in stroke will be developed.

Expected outcome:

By providing a virtually unlimited source of dif-
ferent  types  of  neurons  and  glia,  this  stem  cell 
technology  may  become  the  scientific  break-
through that will render cell replacement a use-
ful treatment strategy for stroke patients. 

Involving  top  European  scientists  with  comple-
mentary  expertise  and  one  SME,  StemStroke, 
using NSCs to repair the stroke-damaged brain, 
can make this research state-of-the-art through 
unique and diverse means: 

•  by demonstrating the capacity of stem 
cells of various sources to expand ex vivo 
and to differentiate into specific neuronal 
phenotypes; 
•  by identifying tools and molecules that 
can  stimulate  endogenous  neurogenesis 
based  on  detailed  knowledge  about  its 
regulatory mechanisms; 
•  by determining whether neurons gen-
erated from NSCs can be anatomically and 
functionally  integrated  into  the  stroke-
damaged brain;  
•  by developing MRI-based imaging for 
monitoring the survival, migration and dif-
ferentiation  of  endogenous,  and  grafted 
NSCs and their progeny, as well as the al-
teration of the lesion in response to stem 
cell therapy; 
•  by showing the level of functional re-
covery after stroke that can be induced by 
grafted and endogenous NSCs; 
•  by  developing  the  first,  scientifically-
based preclinical protocol for the applica-
tion of stem cells in stroke patients.

Strategies for stem cell therapy in stroke patients ex-
plored by StemStroke

The main aim of StemStroke is to develop novel 
strategies to repair the brain and restore function 
after stroke based on the transplantation of NSCs 
or their derivatives, or stimulation of the produc-
tion of new neurons from the adult brain’s own 
NSCs. 

Approach and methodology:

Two main strategies to generate neurons for re-
placement  in  the  stroke-damaged  brain  will  be 
used in StemStroke. In the first strategy, NSCs iso-
lated from human ES cells, or from human foetal 
or adult brain tissue, are expanded in culture, ge-
netically modified (if needed) and subsequently 
transplanted  into  the  recipient  subjected  to 
stroke. In the second strategy, neurogenesis from 
endogenous  NSCs  is  stimulated  using  tools  de-
veloped in the project, leading to the increased 
survival,  migration  or  maturation  of  newly 
formed neurons. 

The morphological and functional integration of 
grafted and endogenously generated NSCs and 
their  progeny  in  the  stroke-damaged  brain,  will 
be  assessed  using  immunocytochemistry,  ana-
tomical tracing techniques and patch-clamp elec-
trophysiology.  A  new  in vivo  MR-based  imaging 
and  behavioural  test  battery,  developed  within 
the proposed project, will be used to assess stem 
cell function and recovery of sensory, motor and 

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REgENERATIvE MEdICINE

Coordinator 

ZaalKokaia
University Hospital
Lund Strategic Research Centre for Stem Cell Biology and 
Cell Therapy
Laboratory of Neural Stem Cell Biology and Neuro-
science Programme, BMC B10
SE-221 84 Lund Sweden 
E-mail: Zaal.Kokaia@med.lu.se

Partners

AustinSmith
University of Cambridge
Wellcome Trust Centre for Stem Cell Research
Cambridge, UK

MathiasHoehn
Max-Planck-Institute for Neurological Research
In-vivo-NMR Laboratory
Cologne, Germany

LilianaMinichiello
European Molecular Biology Laboratory
Mouse Biology Unit
Monterotondo, Rome, Italy 

StephenB.Dunnett
Cardiff University
Brain Repair Group at School of Biosciences
Cardiff, UK

LilianWikström
NeuroNova AB
Stockholm, Sweden

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New Therapies – Regenerative Medicine

63

STEMS

Pre-clinicalevaluationofstemcelltherapyinstroke

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LHSB-CT-2006-037328
SpecificTargetedResearchProject

ECcontribution  e 2400000

Startingdate 1December2006

Duration 36months

Website  http://www.stemsproject.eu

Background and objectives:

The use of stem cells with multipotent properties 
has  become  a  challenging  area  of  research  for 
most clinical fields. It is of particular importance 
in disciplines that desperately lack treatment op-
tions, such as brain disorders and lesions. In this 
context,  stroke  or  ischemic  cerebrovascular  dis-
ease is an important target. Stroke accounts for 
roughly half of the patients hospitalised for neu-
rological diseases, and is associated with a large 
proportion of the healthcare costs in Europe. 

Until  now,  all  neuroprotective  approaches  that 
have  yielded  positive  results  in  animal  models 
of stroke have proved ineffective in clinical trials. 
Given their expected capacity to self-renew and 
differentiate efficiently into the desired cell type, 
clonal populations of stem cells (SC) promise to 
produce beneficial effects in a number of diseas-
es. Several studies already indicate that SC trans-
plantation  has  therapeutic  potential  for  stroke, 
using  embryonic,  foetal  and  adult  SC  sources, 
and lines derived from teratoma. 

ment  at  long-term  post-stroke  delays.  STEMS 
aims  specifically  at  determining  the  extent  and 
limits of SC therapy in stroke, in order to pave the 
way for clinical therapeutic trials. 

Approach and methodology:

To define the therapeutic potential of SC in stroke, 
a  multidisciplinary  study  from  human  stem  cell 
culture  to  the  analysis  of  functional  recovery  in 
animal  models  of  stroke  will  be  implemented. 
The main objectives are to define the following: 
• 
the  most  efficient  cell  source,  by  a 
comparison of embryonic and adult neu-
ral stem cells;
• 
transplantation;
• 
plantation;
the set of behavioural tests and imag-
• 
ing parameters to improve animal model 
predictiveness of functional recovery.

the  window  of  opportunity  for  trans-

ideal  differentiation  stage  for 

the 

Expected outcome:

However, a great deal of crucial information must 
be  found  before  SC  transplantation  becomes  a 
clinical  reality.  For  instance,  the  standardisation 
of the conditions to regulate SC proliferation and 
differentiation  so  as  to  produce  region-specific 
grafts needs to be better defined; and changes in 
the properties of SC, induced by transplantation 
into lesioned brain structures, are poorly under-
stood, as is the full extent of functional improve-

By  addressing  a  fundamental  roadblock  in  the 
development pathway for stroke therapy, STEMS 
will make available a series of validated methods, 
models and analytical tools that will greatly en-
hance the capability of consortium members, as 
well  as  stimulate  the  development  of  new  ave-
nues of research for stroke therapy. The validated 
methods  include  a  culture  of  specific  hESC  and 
hANSC  cell  lines,  up-scaling  and  shipping  con-

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REgENERATIvE MEdICINE

ditions  for  both  cell  types,  and  transplantation 
protocols  in  the  stroke-injured  adult  brain.  The 
validated models will include development of a 
nonhuman primate model of stroke, allowing an 
evaluation of higher cognitive functions. The val-
idated analytical tools will include homogenised 
guidelines  for  functional  evaluation  among  the 
groups,  and  the  development  of  new  MRI  se-
quences for studies in monkeys. 

STEMS will provide the Community with a unique 
set  of  quantified  data  in  stroke  research,  new 
standards,  and  the  proof  of  concept  of  a  new 
therapeutic approach for stroke. It is the consorti-
um’s belief that the results will encourage groups 
of researchers to cooperate, and stimulate indus-
trial interest for the field, so as to rapidly regener-
ate European forces on stroke experimental and 
clinical research. 

Figure A Nestin-positive cells from hESC-derived progeni-
tors transplanted into a rat model of stroke indicate dif-
ferentiation into neural cell types (picture Inserm).

Firgure B Co-localisation of Nestin (red) and human 
nuclear marker (green) identifies the cells as transplanted 
ones (yellow indicates the co-localisation (picture Inserm).

Figure C Magnetic resonance imaging of the brain lesion 
induced by 2 hours occlusion of the middle cerebral 
artery in rats (picture Inserm/CEA) 

New Therapies – Regenerative Medicine

6

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REgENERATIvE MEdICINE

STEMS

Coordinator

BrigitteOnteniente
INSERM Unit 549
Neurobiologie de la Croissance et de la Sénescence 
2 Ter, rue d’Alésia
75014 Paris, France
E-mail: brigitte.onteniente@broca.inserm.fr

EvaSykova
Institute of Experimental Medicine
Academy of Sciences of the Czech Republic
Prague, Czech Republic

ChristianeDascher-Nadel
Inserm Transfert SA
Department of European and International Affairs
Marseille, France

Partners 

PatrikBrundin
Lunds Universitet
Faculty of Medicine
Department of experimental medical science
Lund, Sweden

BenteFinsen
University of Southern Denmark
Medical Biotechnology Centre
Odense, Denmark

JonasFrisen
Karolinska Institute
Department of Cell and Molecular Biology
Stockholm, Sweden

PhilippeHantraye
Centre à l’Energie Atomique
URAD2210 CEA/CNRS: Unité d’imagerie isotopique, 
biochimique et pharmacologique
Orsay, France

JohanHyllner
Cellartis AB
Goteborg, Sweden

KlausReymann
Leibniz-Institut für Neurobiologie
Neuropharmacology Group
Magdeburg, Germany

66

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REgENERATIvE MEdICINE

STROKEMAP

Multipotentadultprogenitorcellstotreatstroke

ContractNo 
Projecttype

LSHB-CT-2006-037186
SpecificTargetedResearchProject

ECcontribution  e 2400000

Startingdate 1October2006
Duration 36months






Background and objectives:

Stroke  is  caused  by  the  occlusion  of  a  cerebral 
artery, resulting in irreversible tissue damage for 
which there is no curative treatment available as 
yet.  Each  year,  approximately  one  million  peo-
ple suffer strokes in the EU. Approximately 25% 
of men and 20% of women experience a stroke 
if they live to 85 years, and stroke is the second 
most common cause of death worldwide.  

STROKEMAP hypothesises that a universal popu-
lation  of  stem  cells  that  can  restore  bloodflow 
through the ischemic region, and that can differ-
entiate  in vivo  into  neurons  and  astrocytes  that 
died due to cerebral ischemia will be the superior 
cell population for the vascular and neural repair 
needed for therapy of stroke. 

The overall hypothesis is that allogeneic Multipo-
tent  Adult  Progenitor  Cells  or  MAPCs,  a  novel 
bone marrow stem cell population first described 
by  the Verfaillie  lab  in  2002,  are  an  ideal  candi-
date stem cell population, and this will be tested 
in STROKEMAP. Indeed, MAPCs can generate ar-
terial  the  endothelial  cells  and  vascular  smooth 
muscle  cells  needed  to  restore  the  vasculature, 
as well as the neural cells and neuroprogenitors 
that could be used to restore neural circuitry.

The objectives of STROKEMAP are:

MAPCs endothelial cells and neural cells;
• 
to  compare  the  efficacy  of  MAPCs  or 
their  differentiated  progeny  with  gold-
standard  cell  populations  (that  is,  bone 
marrow  cells  currently  used  in  animal 
models) to restore circulation and neural 
circuitry damaged by stroke;
• 
to  develop  innovative,  non-invasive 
imaging  techniques  that  will  allow  it  to 
follow cell survival, migration and engraft-
ment  following  transplantation,  as  well 
as  to  gain  insight  into  the  mechanism(s) 
through which stem cells repair the blood 
supply  and  neural  circuitry  in  the  brain, 
and  also  to  provide  quantitative  assess-
ment of such repair;
to  develop  strategies  allowing  trans-
• 
plantation  of  universal  donor  human 
stem  cells,  and  strategies  to  circumvent 
possible immunological rejection;
• 
to  develop  clinical  scale  and  grade 
stem cell populations that could be used 
in  clinical  trials  upon  completion  of  the 
translational  and  preclinical  studies  pro-
posed in this project;
to develop an ethical and legal frame-
• 
work in which to initiate clinical trials with 
stem cells for stroke.

Approach and methodology:

to  carry  out  studies  to  further  un-
• 
derstanding  in  processes  that  underlie 
the  differentiation  and  specialisation  of 

Two  work  packages  will  determine  the  mecha-
nisms  that  underlie  differentiation  of  rodent 
and human MAPC to arterial versus venous en-

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67

 
 
 
 
 
 
 
 
 
REgENERATIvE MEdICINE

STROKEMAP

dothelium and to neural stem cells. This involves 
differentiation in vitro, gene array studies, and for 
the endothelium testing, certain principles in ze-
brafish. 

Another  two  work  packages  are  aimed  at  the 
immunological consequences of grafting MAPC 
in  vivo:  one  evaluating  the  principles  in  mouse, 
and a second where tests will be carried out to 
establish  whether  mice  can  be  given  a  human 
immune  system  using  MAPC.  The  consortium 
will then test the immunological consequences 
of MAPC themselves, or MAPC-derived endothe-
lium  and  neural  cells,  in  the  autologous  and  al-
logeneic setting.

A  fifth  work  package  will  evaluate  whether 
MAPC  can  restore  blood  flow  in  the  brain  and 
neural circuitry. This will be performed chiefly on 
rodents.  A  sixth  work  package  will  evaluate  dif-
ferent methods for labelling stem cells  to allow 
for their visualisation via PET, MRI and BLI, while a 
seventh work package will focus on cell popula-
tions that will be developed, and that conform to 
GMP guidelines. These populations could then be 
used at the end of the granting period in phase I 
or II trials, if the results obtained are encouraging. 
Finally, there is also an ethics work package.

Expected outcome:

The  anticipated  outcome  of  the  STROKEMAP 
project is to:

•  establish  the  possible  beneficial  ef-
fects  of  MAPC  therapy  in  the  setting  of 
stroke; 
•  develop  tools  to  image  the  fate  of 
stem cells in the brain; 
•  understand the immunological conse-
quences of MAPC transplantation; 
•  develop  GMP  procedures  to  expand 
MAPC or their differentiated progeny; 
•  develop  an  ethical  framework  for 
MAPC storage and use.

Coordinator 

CatherineVerfaillie
Stamcelinstituut, K U Leuven
Onderwijs & Navorsing 1 
Herestraat 49 
3000 Leuven, Belgium
E-mail: catherine.verfaillie@med.kuleuven.be

Partners

AernoutLuttun,StefaanVanGool,LucMortelmans,Paul
Schotsmans,BartDeMoor
Katholieke Universiteit Leuven
Leuven, Belgium

PeterCarmelietandMiekeDewerchin
Vlaams Interuniversitair Instituut voor Biotechnology
Leuven, Belgium

FelipeProsperandIvanPenuelas
Universidad de Navarra
Pamplona, Spain

JoséManuelGarcia-Verdugo
Universidad de Valencia
Valencia, Spain

MarkusManz
Institute for Research in Biomedicine
Bellinzona, Switzerland

ErnestArenas
Karolinska Institutet
Stockholm, Sweden

GilVanBokkelen
ReGenesys
Brussels, Belgium

Jean-MarcIdee
Guerbet
Roissy, France

68

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REgENERATIvE MEdICINE

RESCUE

Fromstemcelltechnologytofunctionalrestorationafterspinalcordinjury






ContractNo 
Projecttype

LHSB-CT-200-18233
SpecificTargetedResearchProject

ECcontribution  e 200000

Startingdate 1December200

Duration 36months

Website  www.rescueproject.eu

Background and objectives:

According  to  the  International  Campaign  for 
Cures of spinal cord injury Paralysis (ICCP), more 
than  130  000  people  worldwide  survive  a  trau-
matic  spinal  cord  injury  (SCI)  each  year,  leading 
to permanent paralysis and a lifetime of disabil-
ity.  Thus,  with  an  average  age  at  injury  of  33.4 
years and a nearly normal life expectancy due to 
advances in healthcare, it is clear that the popu-
lation  of  people  with  SCI  is  steadily  increasing 
globally. 

It is estimated that by 2005, over 2.5 million peo-
ple worldwide were living with SCI-induced pa-
ralysis.  In  Europe,  there  are  approximately  330 
000 people suffering from SCI, with more than 10 
000 new cases occurring each year.

SCI has long been regarded as intractable, largely 
due to the alleged inability of the central nervous 
system  (CNS)  to  regenerate.  However,  over  the 
last  two  decades,  technological  advances,  com-
bined with the understanding of the pathophysi-
ology of SCI, have progressed to the point where 
it  is  now  conceivable  to  develop  therapeutic 
intervention  strategies  aimed  at  reconstructing 
the neuronal circuitry damaged by the lesion.

to 

One of the most powerful tools for this objective 
is based on stem cells, which can be used in the 
following ways: 
introduce  permissive  molecules 
• 
and/or  trophic  agents  at  the  level  of  the 
lesion to improve regeneration of the sev-
ered axons; 
to  replace  damaged  cells,  grafted  lo-
• 
cally to stimulate specific circuits such as 
the central pattern generator; 
• 
to  enhance  the  therapeutic  potential 
through  the  activation  of  intrinsic  stem 
cells.

Rescue focuses on the combination of the most 
efficient  technologies  to  direct  the  fate 
of intrinsic, as well as extrinsic stem cells, 
and/or  their  transformation,  in  order  to 
obtain appropriate cell types at the right 
time and in the right place, to promote re-
pair of the injured spinal cord.

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69

 
 
 
 
 
 
REgENERATIvE MEdICINE

of RESCUE can therefore be used as templates for 
the elaboration of therapeutic strategies whose 
application  should  be  broadened  to  other  CNS 
traumatic damages, such as traumatic brain inju-
ries and stroke.

Murine and human adult spinal cord stem cells

RESCUE

Approach and methodology:

RESCUE will investigate a number of different ap-
proaches that are currently available, to try to re-
store function of the injured spinal cord through 
the use of human stem cells from bone marrow 
and the central nervous system. These approach-
es are set out below.

•  Use  of  stem  cells  during  the  acute 
phase  (within  one  week  of  the  injury  in 
man) to reduce inflammation and second-
ary  degeneration  in  the  spinal  cord. This 
also  reduces  the  formation  of  scar  tissue 
which  constitutes  a  major  barrier  to  ax-
onal regrowth.
•  Enhanced  regeneration  of  neurons 
and  axons  in  the  spinal  cord  through  in-
creased  availability  of  axon  growth-per-
missive  molecules  and/or  trophic  agents 
produced  by  stem  cells  or  their  progeny. 
Such molecules may be produced by un-
modified cells, or by cells specifically engi-
neered to do so.
•  Regeneration/restoration  of  function 
by supporting and monitoring the activa-
tion of intrinsic spinal cord stem cells.
•  Restoration  of  function  after  replace-
ment of the damaged cells, following local 
grafting.

Expected outcome:

RESCUE’s  final  objective  is  to  translate  experi-
mental  studies  using  human  stem  cells  in  pre-
clinical animal models into the clinic. This will be 
achieved  through  the  elaboration  of  a  series  of 
therapeutic tools for stem cell therapy to be used 
in a wide variety of clinical paradigms of SCI.

Spinal cord lesions represent an ideal model for 
the  development  of  regenerative  therapy  for 
traumatic lesions of the central nervous system 
(CNS),  as  they  are  more  prone  to  precise  func-
tional characterisation and follow-up than brain 
injuries. Preclinical results obtained in the context 

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REgENERATIvE MEdICINE

JackPrice
Centre for the Cellular Basis of Behaviour
King’s College London
London, UK

Jean-PhilippeHugnot
Université Montpellier 2
Montpellier, France

Coordinator 

AlainPrivat
 INSERM
Unit 583 Physiopathology and treatment of sensorial 
and motor deficiencies
Institut de Neuroscience
Hôpital Saint-Eloi
80 rue Augustin Fliche
34295 Montpellier Cedex 05, France
E-mail: privat@univ-montp2.fr

Partners 

JeanSchoenen
University of Liège
Department of Neurology
Liège, Belgium

EvaSykova
Institute of Experimental Medicine
Academy of Sciences of the Czech Republic
Prague, Czech Republic

JacquesMallet
Centre national de la recherché scientifique - CNRS
Laboratoire Génétique de la Neurotransmission 
Paris, France

ManuelGaviria
Neuréva
Montpellier, France

ChristianeDascher-Nadel
Inserm-Transfert SA
Department of European and International Affaires
Marseille, France

GaryBrook
University Hospital Aachen
Aachen, Germany

MinervaGimenezyRibotta
Consejo Superior de Investigaciones Cientificas
Alicante, Spain

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New Therapies – Regenerative Medicine

71

STEM-Hd

Stem cells for therapeutics and exploration of mechanisms in Huntington’s
disease

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LHSB-CT-2006-037349
SpecificTargetedResearchProject

ECcontribution  e 200000

Startingdate 1December2006

Duration 36months

Website  www.stemhd.eu

Background and objectives:

Huntington’s  Disease  (HD)  is  a  rare  monogenic 
disorder. Age of onset of clinical symptoms of HD 
is  quite  variable,  although  the  disease  starts,  in 
the  majority  of  cases,  at  adulthood  (average  35 
years of age). Pathological lesion primarily affects 
the GABAergic output medium-spiny neurons of 
the  striatum.  Clinical  symptoms  are  character-
ised by a rapidly progressive alteration of motor 
abilities (chorea, bradykinesia), psychological dis-
turbances (depression and irritability) and cogni-
tive impairment (of most functions, by the later 
stages), leading to dementia. On average, death 
occurs 15 to 20 years later. 

HD  is  an  autosomal,  dominant  monogenic  dis-
ease. The mutation consists of an enlarged CAG 
triplet repeat that encodes an elongated stretch 
of polyglutamine in the N-terminal portion of the 
large protein huntingtin. Neither the physiologi-
cal role of huntingtin, nor the molecular mecha-
nisms of the pathology are currently known, but 
since its discovery, a number of cellular pathways, 
interacting proteins and nucleic acids have been 
identified.

HD research has benefited from a direct genetic 
screen  for  more  than  12  years,  and  preimplan-
tation  genetic  diagnosis  has  been  proposed  to 
at-risk  couples  for  more  than  6  years.  However, 
no  validated  treatment  —  neither  curative  nor 
symptomatic — is available for HD today.
The fundamental objective of STEM-HD is to con-

tribute in a decisive manner to the understand-
ing  of  the  mechanisms  of  HD,  as  a  necessary 
step toward finding a cure. The consortium aims 
at  reaching  two  main  complementary  goals:  to 
decipher  molecular  mechanisms  of  HD,  and  to 
identify  compounds  endowed  with  therapeutic 
potential for HD.

Approach and methodology:

To realise its goals, STEM-HD will perform the fol-
lowing activities: 

•  use  an  available  human  embryonic 
stem (ES) cell line, based upon the hypoth-
esis that ES cells expressing a disease-re-
lated  mutant  gene  may  be  used  for  mo-
lecular  modelling  of  that  monogenic 
disease; 
implement  large-scale  technological 
• 
resources,  and  combine  resource-driven 
and hypothesis-driven analyses at all stag-
es of the project. 

Expected outcome:

The identification of new biomarkers and of the 
molecular  pathway  of  HD  may  lead  to  possible 
therapeutic applications, and the development of 
new diagnostic tools. These tools will allow spe-
cialists to follow the development of the disease 
in  patients  known  as  carriers  of  the  mutation, 
helping them to determine the best time to start 
the palliative treatments (psychological care, ex-
ercise, etc), or the treatments, once developed. It 

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REgENERATIvE MEdICINE

Figure A Colony of undifferentiated human embryonic 
stem cells (200X, passage 19) on a monolayer of feeder 
cells. hES marker proteins Oct4 and Nanog stained in red 
and green, respectively; nuclear staining in blue (DAPI).

Figure B hES cells differentiated into neuronal progeni-
tor cells (day 45).  Immature neural cells stained in red 
(Nestin), young neurons in green (Tuj1); nuclear staining 
in blue (DAPI).

will also be of help to facilitate the identification 
of the disease in patients not known as carriers of 
the mutation. Finally, it will be a valuable tool to 
evaluate in vitro in patients, the effectiveness of 
any new treatment in clinical trials for HD.

Although  the  focus  of  the  consortium  will  be 
dedicated fully to HD, the participants’ common 
goal is to extend the value of their achievements 
by making it a model disease for designing pro-
tocols  and  infrastructures  applicable  to  many 
other  monogenic  diseases.  Because  each  rare 
monogenic disease affects only a limited number 
of people (less than 5 in 10 000), it has been dif-
ficult until now to allocate to each affected indi-
vidual,  the  investments  required  by  large-scale 
approaches,  that  are  used  for  non-rare  diseases 
both for the exploration of mechanisms and for 
high throughput/high content drug screening. 

The  fact  that  these  monogenic  diseases  are  di-
verse in terms of their phenotype, and that each 
one is relatively rare, have made them compara-
tively  unattractive  targets  for  drug  discovery. 
STEM-HD    aims  at  directly  addressing  these  is-
sues,  by  reducing  the  specificity  of  the  process 
for each disease — through the standardisation 
of common protocols and infrastructures for all 

Figure C Mature GABAergic neurons (day 63 of dif-
ferentiation). GABAergic striatal neurons stained in red 
(DARPP-32), neuronal cells in green (MAP2); nuclear stain-
ing in blue (DAPI).
© Photos Inserm

— and by restricting its workload to a minimum 
through  systematic  automation,  thus  providing 
the opportunity to search for therapeutics of sev-
eral rare monogenic diseases.

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New Therapies – Regenerative Medicine

73

REgENERATIvE MEdICINE

ChristianeDascher-Nadel
Inserm-Transfert SA
Department of European and International Affairs
Marseille, France

STEM-Hd

Coordinator

MarcPeschanski
INSERM Unit 421, I-STEM
BP 118, 1 rue de l’Internationale 
91004 Evry cedex, France
E-mail: mpeschanski@istem.genethon.fr

Partners 

JosephItskovitz-Eldor
Technion - Israel Institute of Technology
Stem Cell Centre, Faculty of Medicine
Haifa, Israel

DoroteaRigamonti
Dialectica srl
Nerviano, Italy

JacquesHaiech
Université Louis Pasteur Strasbourg
Faculté de Pharmacie
LC1 UMR7175 - Groupe Chimiogénomique et Pharma-
cogénomique
Illkirch, France

NicholasAllen
Cardiff University
School of Biosciences
Cardiff, UK

ElenaCattaneo
Universita degli Studi di Milano
Department of Pharmacological Sciences and Centre of 
Excellence on Neurodegenerative Diseases
Laboratory of Stem Cell Biology and Pharmacology of 
Neurodegenerative Disorders
Milan, Italy

KarenSermon
Vrije Universiteit Brussel
Research Group Reproduction and Genetics
Brussels, Belgium

74

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REgENERATIvE MEdICINE

NEUROSCREEN

Thediscoveryoffutureneuro-therapeuticmolecules

ContractNo 
Projecttype

LHSB-CT-2006-0300
SME-SpecificTargetedResearchProject

ECcontribution  e 287100

Startingdate 1March2007
Duration 36months






Background and objectives:

NEUROscreen is an industry-led project combin-
ing  novel  neural  stem  cell  bioassays  and  post-
genomic  chemical  genetics,  so  as  to  discover 
innovative therapies in the fields ofneurological 
diseases, regenerative medicine and cancer. A key 
objective is to join fledgling and well-established 
European  commercial  partners  in  a  pre-com-
petitive, collective drive to translate fundamental 
stem cell biology into a state-of-the-art discovery 
platform,  and  successfully  demonstrate  the  po-
tential exploitability of the concept while simul-
taneously strengthening European bio-industry. 

Approach and methodology:

The  project  work  plan  consists  of  6  interlinked 
work  packages  (WPs). WP1  focuses  on  the  pro-
curement,  derivation,  banking,  annotation  and 
establishment  of  quality  control  standards  for 
a  range  of  rodent  and  human  neural  stem  cell 
lines, including brain stem cell tumour lines. WP2 
is the bioassay design component covering the 
development  of  key  genetic  modification  tech-
nology (BAC) and the establishment of a reper-
toire of lines containing reporter constructs. WP2 
will  provide  a  valuable  chemical ‘hit’  validation 
platform  involving  the  verification  of  protein 
function using RNA interference. 

Using modified cell lines to design bioassays re-
quires a number of key validatory steps to be per-
formed in order to assess the integrity of the cells. 

Novel neural stem (NS) cells stained with antibodies 
specific for a defining marker of the cells

WP3 and WP4  provide essential confirmation of 
specification potential for cell lines in use, via in 
vitro (WP3) and in vivo (organotypic slice culture; 
WP4) assessment. WP5 focuses on the provision 
of stem cell lines of adequate quality and quan-
tity  for  screening,  as  well  as  the  compilation  of 
technology  components  and  processes  for  the 
automated  handling  of  cells  for  the  screening 
activity.  Cell  formatting  will  be  the  precursor  to 
the screening activities in WP6. A two-site, two-
platform  approach  will  be  used  for  the  screens 
using a large number of chemicals pre-selected 
by in silico-based design. Leads will be validated 
through WP2, WP3 and WP4 activities.

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REgENERATIvE MEdICINE

NEUROSCREEN

Expected outcome:

NEUROscreen will present a detailed description 
of  the  comparative  properties  of  human  neural 
stem  cells  derived  either  from  tumor  or  normal 
biopsies, and will offer a comparative fidelity of 
functional properties of neural stem cell-derived 
neurons,  versus  those  isolated  acutely  ex  vivo. 
Procedures for the automated expansion of sta-
ble, proliferating human neural stem cells, as well 
a repertoire of bioassays to measure differentia-
tion and subtype specification from neural stem 
and progenitor cells, will be set up. The NEURO-
screen project will establish a repository of anno-
tated stem cell lines, with product release criteria 
based on defined QA/QC characteristics, as well 
as a defined set of bioactive, optimal compounds 
to be validated as contenders for prospective re-
generative medicines.

Coordinator

TimAllsopp
Stem Cell Sciences Plc
Minerva Building 250, Babraham Research Campus
Babraham, Cambridge, CB22 4AT, UK
E-mail: Tim.allsopp@stemcellsciences.com

Scientific coordinator

LilianHook
European Research Programme Manager
Stem Cell Sciences UK Ltd
Roger Land Building
Kings Buildings
West Mains Rd
Edinburgh, EH9 3JQ, UK 
E-mail: Lilian.hook@stemcellsciences.com

Partners

DoroteaRigamonti
Dialectica s.r.l.
Milan, Italy

PasqualeDeBlasio
BioRep s.r.l.
Milan, Italy

StefanieTerstegge
Life & Brain GmbH
Biomedizinische & Neurowissenschaftliche
 Technologie-Plattform
Bonn, Germany

AustinSmith
University of Cambridge
The Wellcome Trust Centre for Stem Cell Research
Cambridge, UK

LucianoConti
Università Degli Studi di Milano
Dipartimento di Scienza Farmacologiche
Milan, Italy

OliverBrüstle
University of Bonn
Institute of Reconstructive Neurobiology
Bonn, Germany

FrançoisGuillemot
National Institute Medical Research
Molecular Neurobiology Division
London, UK

CesareSpadoni
Albany Medical Research Inc
(formerly ComGenex)
Budapest, Hungary

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REgENERATIvE MEdICINE

MYOAMP

Amplificationofhumanmyogenicstemcellsinclinicalconditions

ContractNo 
Projecttype

LSHB-CT-2006-037479
SME-SpecificTargetedResearchProject

ECcontribution  e 2480000

Startingdate 1December2006

Duration 36months






Background and objectives:

Many groups have used animal models to inves-
tigate  the  possibilities  of  using  autologous  cell 
therapy for muscular dystrophies, but these data 
are  dispersed,  not  always  comparable  and  little 
attention has been focused on the transfer of this 
knowledge towards applications for therapeutic 
trials. 

Data exist on injecting murine cells into mouse 
muscle,  but  information  regarding  human  cells 
is sparse. The feasibility of autologous myoblast 
transfer therapy has already been demonstrated 
for  cardiac  repair,  even  if  in  cardiac  therapy,  in-
jected cells were mainly used to counteract the 
development of fibrosis in patients devoid of any 
defect in skeletal muscle. 

The fact that preclinical trials developed in mouse 
models of muscular dystrophies have been suc-
cessful as compared to clinical trials, which used 
mostly allogenic cells and resulted in very limited 
clinical benefit for the patients, illustrates the ur-
gent  need  for  preclinical  studies  using  human 
cells.

Myoamp  will  aim  at  defining  conditions  and 
guidelines  to  produce  transduced  human  stem 
cells as vectors for clinical trials. Myoamp will syn-
ergise expertises from European leaders in their 
respective  field  to  set  up  conditions  for  autolo-
gous transfer of human stem cells in GMP condi-
tions for the treatment of DMD by exon-skipping. 

It will ensure that these conditions and guidelines 
are transferred to SME and clinicians, defining ef-
ficient  integration  through  dedicated  partners 
within the 3-years duration of this project. 

Many clinical trials using muscle cells have been 
developed  in  the  past  for  Duchenne  Muscular 
Dystrophy with very limited success. The recent 
emergence  of  new  therapeutic  venues,  based 
upon  post-transcriptional  genetic  corrections 
called “exon-skipping”, have raised new hope for 
this disease. Using viral transfer approaches it has 
given  very  promising  results  but  cannot  reach 
every muscle of the body and trigger a immune 
response to the vector. Autologous cell therapy 
may bypass this reaction and be used as a com-
plement  or  alternative  if  the  cell  type  used  ful-
filled both being an efficient vector and bringing 
a functional benefit to the diseased muscle.

Autologous  muscle  cells  cannot  be  used  since 
these  are  already  defective  in  dystrophic  mus-
cle, while stem cells from other origins are ideal 
candidates,  as  long  as  their  myogenic  and  pro-
liferative potentials are ensured. In this perspec-
tive  mesoangioblasts,  which  have  already  been 
used  in  a  mouse  model  of  muscular  dystrophy, 
and  AC133  cells  have  a  therapeutic  potential 
as demonstrated in the mouse, but very little is 
known about the conditions required to amplify 
in GMP conditions these stem cells isolated from 
humans, which is an essential step required be-
fore any clinical trial.

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In addition to basic knowledge on the amplifica-
tion mechanisms, myoamp will bring guidelines 
and  standard  operative  procedures  to  obtain 
these  cells  in  a  reproducible  and  safe  manner, 
that can be directly transferred to SMEs and clini-
cians for clinical applications. 
These guidelines will therefore address technical, 
ethical and safety issues in a GMP environment. 

Potential applications:

•  preclinical  protocols,  standard  oper-
ating  procedures  to  characterise,  amplify 
and  assess  myogenic  human  stem  cells 
for autologous cell therapy treatments in 
muscle disease. 
•  ethical and safety procedures to cover 
the protocols.
• 
set of growth factors.

specific culture medium with a defined 

MYOAMP

Myoamp will address the question of the amplifi-
cation of these cells used as autologous cell ther-
apy vectors and their safety. The cells transduced 
with a lentiviral construct allowing exon-skipping 
will be selected and further amplified. It should 
be noted that transduced cells may be cloned so 
that their integration site is determined), as will 
be proposed in the part of myoamp that focus on 
safety and ethics. In vitro and in vivo approaches 
will  bring  understanding  on  the  regulation  of 
proliferation, while the telomere length (reflect-
ing  the  mitotic  clock  status  of  the  cell)  will  be 
monitored, as well as a combination of receptors 
known to trigger proliferation, (FGF, IGF1, ). 

The stability of the parameters initially examined 
in non-GMP conditions, will be checked through 
amplification in various conditions, to allow the 
definition of both guidelines for GMP production 
and  key-parameters  to  be  followed  during  the 
GMP amplification. 
The number of cells to be injected at each implan-
tation, which is purely empirical in many clinical 
trials, will be tested and optimised in a model of 
implantation of human cells in immuno-deficient 
mice, in order to define the maximum number of 
cells to be finally amplified in GMP conditions.

Role of SMEs

1.  3H  Biomedical  cell  provider  and  re-
sponsible  for  defining  SOP  for  cell  han-
dling;
2.  CELLGENIX  development  of  adapted 
serum-free culture medium and definition 
of SOP; 
3.  GENOSAFE development of safety pro-
cedures and assays for the process.

Expected results: 

The  main  expected  result  is  to  obtain  the  final 
product, i.e. protocols to obtain amplified human 
stem cells, in a state that will allow an optimised 
efficiency in injections in vivo. 

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REgENERATIvE MEdICINE

AntonOttavi
Inserm-Transfert
Paris, France

Scientific coordinator 

VincentMouly
INSERM 
UMR S 787 Myologie
Institut de Myologie
105 bd de l’Hôpital
75634 Paris Cedex 13, France
E-mail: mouly@ext.jussieu.fr

Partners

LuisGarcia
INSERM
Paris, France

YvanTorrente
University of Milan
Milan, Italy

GiulioCossu
Fondazione Centro San Raffaele del Monte Tabor  
Milan, Italy

JennyMorgan
Imperial College, London
London, UK

OttoMerten
Généthon, 
Evry, France

MallenHuang
3H Biomedical
Uppsala, Sweden

RolandBosse
Cellgenix Gmbh, 
Freiburg, Germany

VincentGiuliani
Genosafe SA
Evry, France

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79

CRYSTAL

Cryo-bankingofstemcellsforhumantherapeuticapplication

REgENERATIvE MEdICINE






ContractNo 
Projecttype

LSHB-CT-2006-037261
SpecificTargetedResearchProject

ECcontribution  e 2400000

Startingdate 1February2007
Duration 36months

Website  www.crystal-eu.org

Background and objectives: 

Stem cells are currently at the centre of biomedi-
cal research. Besides advancing the basic under-
standing  of  the  human  development  and  the 
cellular differentiation processes, stem cells hold 
the  unique  potential  for  novel  therapies  of  de-
generative diseases such as ischemia of the heart, 
Parkinson’s disease, diabetes, and certain types of 
tumours. 

Future  human  stem  cell  therapy,  however,  will 
have to build on a readily available, safe and reli-
able supply of high-quality human stem cells that 
must be assured by cell banking. Current banking 
approaches  still  rely  on  storing  sources  of  stem 
cells rather than on the banking of defined, well-
characterised  stem  cell  populations  (both  adult 
and embryonic). It should be noted that there is 
room for improvement of the conditions for reli-
able outgrowth, since they are still at a rudimen-
tary stage. The isolation, identification and culture 
of stem cells are not standardised among labora-
tories and reproducibility of protocols is limited. 

As  the  culture  of  human  embryonic  stem  cells 
routinely requires the use of animal products or 
cells, their therapeutic use is ruled out. In vitro cul-
ture and expansion of haematopoietic stem cells, 
either from adult bone marrow or cord blood, is 
far from optimal, necessitating further research in 
order to overcome problems related to the insuf-
ficient  numbers  of  obtained  stem  cells  and  the 
ageing of the stem cell population. 

In addition, for many stem cell types cryopreser-
vation itself is neither optimised nor validated for 
different cell types, and there are multiple cellu-
lar  and  biophysical  challenges  to  be  addressed, 
in  order  to  define  optimised  cryopreservation 
protocols. Current methods represent a trade-off 
between preventing formation of damaging ice 
crystals  and  toxic  effects  of  cryoprotectants.  In 
particular, the amenability of different stem cell 
populations  to  freezing  conditions  is  not  well 
understood.  The  viability  of  human  embryonic 
stem  cells  is  low  after  freezing,  and  short-  and 
mid-term  effects  of  freezing  on  cellular  proper-
ties remain to be investigated.

The aim of CRYSTAL is to develop tools and pro-
cedures to enable the cryopreservation of differ-
ent stem cell types for the generation of sufficient 
numbers of high-quality cells suitable for safe hu-
man stem cell therapy. To this end, CRYSTAL will 
conduct focused research on the methods, tools 
and protocols required for optimal cryopreserva-
tion and banking of stem cells. 

In  order  to  achieve  this  objective,  the  following 
unresolved  methodological  and  experimental 
aspects of stem cell banking will be addressed:

scientific  validation  and  optimisation 
• 
of  protocols  for  identification,  charac-
terisation, maintenance and expansion of 
stem cells;
•  establishment  and  validation  of  the 
cryopreservation of stem cells; 
•  definition of validation methods of the 

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REgENERATIvE MEdICINE

properties  of  stem  cells  after  defreezing, 
including  maintenance  and  expansion, 
engraftment capability, etc.

Approach and methodology: 

The  CRYSTAL  project  pursues  an  integrated  ap-
proach, using both somatic and embryonic stem 
cells to address existing shortcomings that limit 
the routine application of stem cell banking with 
a  therapeutic  perspective.  The  consortium  will 
develop  tools  and  optimised  procedures  to  en-
able cryopreservation of different stem cell types 
and  allow  for  the  safe  production  of  sufficient 
numbers  of  high-quality  cells  for  future  human 
therapy.  This  will  comprise  standardised  proto-
cols  and  tools  for  stem  cell  isolation,  identifica-
tion and culture, novel approaches to their cryo-
preservation (e.g. novel cryoprotectants, freezing 
in  different  conformations)  and  an  automated 
quality control system for stem cell preparations. 

Five  stem  cell  research  laboratories  providing 
four different sources of adult (from cord blood, 
bone marrow and placenta) and human embry-
onic  stem  cells  have  teamed  up  with  two  part-
ners  specialising  in  applied  banking  and  fun-
damental  cryobiological  research.  The  scientific 
experimental  work  is  supported  by  a  unit  that 
will create a common knowledge base for inte-
grating pre-existing know-how from both inside 
and  outside  the  consortium,  and  for  guiding 
partner laboratories in the implementation and 
refinement of standard operating procedures for 
culturing techniques, cryopreservation and vali-
dation of protocols. 

The scientific work is to be supported by profes-
sional  project  management  and  a  team  ensur-
ing  the  effective  dissemination  and  use  of  the 
obtained  results.  CRYSTAL  is  thus  in  a  position 
to  solve  existing  problems  using  an  integrated, 
systematic  approach,  and  to  provide  standard-
ised, reproducible methods and tools to advance 
therapeutic stem cell research in Europe. 

Figure (© IBMT): Cryopreservation issues and novel ap-
proaches to cryopreservation.

Fig. 1 - combined block face scanning electron microscopy 
(back scattered mode) with freeze substitution of cryopre-
served adherent murine fibroblasts. The arrow indicates an 
intracellular ice domain.

Fig. 2 - scanning electron micrograph (secondary electron 
mode) of adherent cells (murine fibroblasts, gold) on carbon 
based nanostructured fibres (blue). The image is manually 
colored, horizontal image width about 120 microns. 

Expected outcome: 

CRYSTAL plans to deliver a set of optimised, vali-
dated protocols, covering the core aspect of stem 
cell banking, and achieving significant innovation 
in the three areas of preparation and cultivation 
methods,  preservation  methods  and  validation 
methods.  These  optimised,  validated  methods 
and tools will be made available to the scientific 
community  to  underpin  initiatives  on  stem  cell 
banking,  thus  providing  a  solid  foundation  for 
the future development of stem cell therapies. 

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81

CRYSTAL

Coordinator

JürgenHescheler
University of Cologne / Clinical Centre of the University 
of Cologne
Institute of Neurophysiology
Robert-Koch-Str. 39
50931 Cologne, Germany
E-mail: J.Hescheler@uni-koeln.de

Partners 

GuyWouters
Life-Sciences Group N.V. 
HB Zutphen, Netherlands

HeikoZimmermann
Fraunhofer Institute for Biomedical Engineering (IBMT)
St. Ingbert, Germany

AndreaKolbus
Medical University of Vienna
Department of Obstetrics and Gynaecology
Vienna, Austria

AndreasZisch
University Hospital Zurich
Department of Obstetrics
Zurich, Switzerland

CatherineVerfaillie
Katholieke Universiteit Leuven
Interdepartementaal Stamcelinstituut
Leuven, Belgium

PeterPonsaerts
University of Antwerp
Laboratory of Experimental Hematology
Antwerp, Belgium

AnnetteRingwald
ARTTIC
Paris, France

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gENE THERAPY

84

New Therapies – Gene Therapy

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gENE THERAPY

CLINIgENE

Europeannetworkfortheadvancementofclinicalgenetransferandtherapy






LSHB-CT-2004-018933
ContractNo 
Projecttype NetworkofExcellence

ECcontribution  e 12000000
Startingdate 1April2006

Duration 60months

Website  www.clinigene.eu

Background and objectives:

CLINIGENE aims to: 

As the field of gene therapy has matured, oppor-
tunities  have  been  created  that  are  both  excit-
ing  and  promising,  and  some  treatments  have 
already been proven clinically effective. However, 
precise  quality  and  safety  standards  for  clinical 
gene transfer have yet to be defined. 
In this context, establishing optimal methods for 
the production of both standard and innovative 
vector systems would pave the way for acceler-
ated product development and improved safety. 
This  would  be  of  enormous  value  to  patients, 
individual investigators, industry and regulatory 
authorities alike.

The  move  from  the  preclinical  to  the  clinical 
phase  calls  for  clinical-quality  grade  products 
manufactured  for  human  use.  In  addition,  the 
safety profile of these products must ensure that 
the  potential  benefits  outweigh  potential  side 
effects,  thus  providing  patients  with  a  safe  and 
efficacious treatment option. 
The role of CLINIGENE is to efficiently mobilise all 
interested  parties,  involving  academic  research 
and  production  centres  in  particular,  alongside 
companies, patients’ groups and regulatory bod-
ies.  Its  goal  is  to  integrate  multidisciplinary  re-
search  so  as  to  decipher  the  key  elements  that 
can lead to improved safety and clinical efficacy 
of  gene  transfer/therapy  products.  Control  and 
test methods will be established, and can then be 
applied as platforms for particular gene transfer 
products. 

foster  interaction  between  all  stake-
• 
holders: regulators, preclinical and clinical 
investigators,  scientists,  companies  (oth-
erwise  competitors),  as  well  as  patients’ 
groups, in order to streamline integration 
of multidisciplinary expertise;
•  establish  quality,  safety,  efficacy  and 
morally  acceptable  standards  for  clinical 
gene transfer products;
identify the ‘critical path’ to accelerate 
• 
the  transit  phase  from  the  preclinical  to 
the clinical phase by integrating expertise 
and generating new knowledge;
improve European competitiveness by 
• 
spreading  excellence  and  disseminating 
knowledge;
•  gain  a  clinically  significant  improve-
ment in the treatment of human disease 
through gene therapy.

Approach and methodology:

The network initially analysed the bottlenecks that 
exist. From this assessment, partners were able to 
better understand current and future limitations, 
and could then examine and offer potential solu-
tions and methods (Table 1).
The  CLINIGENE  project  will  not  establish  stand-
ards  in  retrospect,  based  on  previous  trials;  it 
will rather augment the development of entirely 
novel  protocols  using  cutting-edge  technology. 
Practical  examples  of  new  knowledge  applica-
tions  and,  in  the  longer  term,  of  clinical  success 

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8

 
 
 
 
 
 
 
 
gENE THERAPY

CLINIgENE

Technological 
platforms

Bottlenecks
to be addressed

Current
limitations

AAV

• Serotype production

Adenovirus

• Gutless vector 

production
•	Efficiency	of	

oncolytic Ad-vectors 
•	Non	specific	uptake/	

Targeting

•  Reproducibility
•  Titers
•  Scale up
•	 Non-specific	
encapsidation

•	 Lack	of	

standardized high 
yield production 
system
•	 Unspecific	

interaction of 
vector with non-
target cellular 
and extracellular 
compartments
•  Contamination by 

helper virus
•  Low degree of 
Tumour spread

Retrovirus

• Integration
• Non-characterized 
packaging	cell-lines

• Targeting

• Vector titers
•	Purification
• Stability

Lentivirus
Besides 
Retroviruses

•  Safety issues
•  Partial recombination
•  Validation of RCL assays
•	 Stable	packaging	cell-lines	
and toxicity of viral proteins

•  Lentivirus associated 

tumour development in liver 
•  Exclusion of regulatory viral 

genes (rev sequences)

•  Quality of vector 

preparation for in vivo use

•  Mode of 

production (stable 
versus	transient),	
purification	and	
quality
•  Low-yield
•  Plasmid 

contaminants

•  Synthesis of viral 
proteins by target 
cells

Solutions to propose 
to be tested & if 
possible exemplified 
by the NoE

• Stable cell-lines 

for each selected 
serotype

•  Establish SOPs and 
improved monitoring 
methods to achieve 
improved production and 
purification	

•  Improved production cell 

lines

•	 Capsid	modification/

polymer	coating/	specific	
targeting

•  Genome optimisation 

(Oncolytic)

•	 Characterized	packaging	

cell-lines

•	 Packaging	cells	with	

specific	integration	sites

•	 Novel	purification,	

formulation & storage 
improving titers

•  Pseudotyping with a 

variety of virus envelopes
•  Locus insultators in target 

cells

•	 Specific	integration

• Chromatography 
based	purification

• Test methods & 

reference material 
for replication 
competent 
recombinant

Cell Therapy
(gene-
manipulated 
cells)

• Differentiation
• Long-term function
•	Immune	attack	to	

the cells 

•  Homogeneity of 

source & phenotype 

•	 Viability/Survival
•  Distribution and 

Homing

•  Testing methods for 
homing & distribution

•	 Cell-marking
•  Optimised culture 

conditions

Non-viral 
technologies
General

• Bacterial sequences 

(CpG,	selection	
genes)

• Mitotic stability
• Replicating 
episomes

•	SMAR/ORI
• Minicircles

No vectorisation
Naked DNA

• Limitation to a few 
specific	situations

•	Efficacy

Physical 
vectorology 
Electroporation

• Safety
• Perception of the 

technology (fears of 
the electricity)

Chemical 
vectorology Present 
DNA formulations

•  Purity of the products
•  Too large number of 

options

•	 Lack	of	standards
•	 Lack	of	

comparisons

•	 Specific	electrodes

•  Control algorithms

•	 SOP	preparation,	

evaluation,	diffusion

•  Training

• Reference 
materials

Other bottlenecks
(not addressed by 
CLINIGENE)

•  Size of the insert
•  Intracellular barriers
•  Route of administration
•  Vector targeting

• Free access to 
   transcomple-

mentation cell-
lines

•  Insertional 

mutageneis in target 
cells

•  Vector titers
•  Size of transgenic 

sequences
•  Number and 

distribution of vector 
integration sites per 
cell

• Number and 
distribution of 
vector integration 
sites per cell

•  Cell-transformation
•  Heterogeneity of 

autologous primary cells

•  Adventitious viruses

•  Acute toxicity of 
delivery method

•  Potential genotoxicity 
of physicochemical 
methods (e.g. DNA 
damage due to electric 
forces in electroporation)

•  Standard procedures 

for material preparation

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gENE THERAPY

are  to  be  provided. The  network  will 
address  orphan  diseases,  such  as 
Fanconi’s  anaemia  or  Duchenne’s,  as 
well  as  more  prevalent  pathologies, 
including  cancer,  cardiovascular  and 
neurodegenerative disorders.
Common standards of reference must 
be shared in order to establish qual-
ity.  Firstly,  such  standards  must  be 
tested,  broad  consensus  must  then 
be reached and subsequently agreed 
upon by the academic centres and the 
companies specialising in the manu-
facture  of  gene  transfer  vectors  for 
the purpose of human gene therapy. 
Due to the diversity of the emerging 
needs,  the  standards  will  be  worked 
out along distinct timelines from one 
vector system to the next, during the 
course of the programme (Fig 1a&b). 

is  developing  platform 
CLINIGENE 
databases  for  particular  vectors  with 
respect  to  their  safety,  efficacy  and 
quality standards in order to acceler-
ate  the  transition  from  research  to 
clinical trials (Fig 1a&b). 
Safety  will  be  considered  both  in 
terms  of  pharmaco-toxicology  and 
viral safety. Whatever the technology, viral safety 
assessment  includes  a  series  of  common  check-
points to consider and also standardise. Another 
important  safety  issue  relates  to  vector  integra-
tion  with  a  concurrent  risk  of  insertional  muta-
genesis. The EMEA/CHMP-GTEG (now GTWP) has 
reported on serious adverse events in clinical trials 
involving X-linked severe combined immunodefi-
ciency  patients.  More  work  is  needed  on  a  case-
by-case basis in order to evaluate the risks accord-
ing to the nature/function of the gene of interest 
and the disease to be treated. The network is ad-
dressing potential improvements, using a variety 
of  experimental  approaches.  CLINIGENE  recently 
submitted  its  comments  to  the  FDA-Long  Term 
Follow-Up guideline, the relevance of which is rec-

Figure 1a & 1b: The European Network Structure and 
outcomes

ognised by regulatory authorities.
Pharmaco-toxicology profiles can be drawn only 
in a case-by-case manner, combining the vector 
system of choice and the specific gene address-
ing the disease in question. A challenge for the 
network  is  to  successfully  mobilise  partner  ex-
pertise in order to obtain useful clinical  trial in-
formation, which will form the basis on an inter-
national effort to use the existing technology for 
the treatment of rare diseases in particular. Prac-
tical clinical protocols will be considered, dealing 
mainly  with  the  definition  of  the  requirements 
for patients’ clinical monitoring. These include: 

thorough definitions of biological sam-
• 
ples to be taken and of those to be stored 
where retrospective studies are required; 

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87

 
 
CLINIgENE

gENE THERAPY

Fig. 2

•  points  to  be  considered  and  steps  to 
be  taken  towards  the  long-term  follow-
up of patients, in particular those patients 
treated with integrating vectors at risk for 
insertional mutagenesis.

The  CLINIGENE  network  has  adopted  an  inte-
grated approach targeting high-level education, 
training  and  human  mobility  of  post-doctoral 
scientists,  doctors  and  pharmacists  in  order  to 
support  a  European  dimension  of  highly  spe-
cialised  researchers,  health  professionals  and 
managers.  The  clinical  development  process  is 
expanded  with  this  approach  in  collaboration 
with the European Society of Cell & Gene Thera-
py. Contacts have already been established with 
other  EC-funded  gene  therapy-related  research 

programmes to streamline innovation and opti-
mise dissemination (Fig 2). 

To successfully integrate its efforts — given the 
breadth of the technological approaches and ap-
plications  anticipated  within  CLINIGENE  —  the 
network coordinator has developed the innova-
tive  scientific  and  strategic  management  soft-
ware  called  Clinisoft,  accessible  online  through 
a  confidential  and  restricted-access  website.  It 
serves several purposes, and functions in the fol-
lowing ways:

•  a  virtual  and  highly  hierarchic  work-
space  supporting  information  exchange 
and collaborative work on documents;
•  an  archive  for  all  important  network-
produced information and documents; 

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gENE THERAPY

•  an  online  coordination,  planning  and 
network monitoring tool; and
•  a direct communication tool between 
defined  working  groups  or  technology 
platforms.

Expected outcome:

The partnership will combine its efforts and skills 
to  initiate  clinical  approaches  and  decipher  the 
key  elements  that  can  lead  to  clinical  success. 
Implementing  benchmarking  activities  will  ef-
fectively  establish  good  practices  as  well.  In  or-
der  to  promote  safe  and  high  quality  clinical 
gene  transfer  treatments,  CLINIGENE  will  apply 
a series of imaging technologies in collaboration 
with the DiMI FP6 NoE on Molecular Imaging and 
Industry partners. CLINIGENE aims to achieve the 
following: 

conducting of phase I trials addressing 

• 
previously-unasked questions; 
•  prevention  of  failures  at  the  clinical 
stage,  as  they  would  have  negative  con-
sequences for patients entering the study 
and would penalise the field in general; 
•  delivery of practical results that would 
open  new  opportunities  for  funding  re-
search  and  clinical  development  in  this 
field, as well as promoting the expansion 
of the high-tech industrial sector.

In developing the actions initiated by the Eure-
genethy2 network, CLINIGENE seeks to reinforce 
links  to  the  international  community.  The  net-
work  will  address  NIH/OBA-RAC  and  FDA-CBER, 
contribute  to  the  International  Liaison  Group, 
and  promote  interaction  through  the  interna-
tional  committee  of  the  American  Society  of 
Gene  therapy.  By  encouraging  and  favouring 
contacts,  cooperation  and  data  sharing  with 
members  of  the  international  community,  such 
as  the  USA  and  Japan,  CLINIGENE  will  also  ad-
vance the strengthening and creation of jobs in 
the biotechnology sector.

Coordinator

OdileCohen-Haguenauer
École Normale Supérieure de Cachan & AP-HP
Laboratoire de Biotechnologies et Pharmacologie géné-
tique Appliquée (LBPA) 
61, avenue du President Wilson
94235, Cachan Cedex, France
E-mail: clinigene.noe@lbpa.ens-cachan.fr

Partners

AlbertoAurricchio
TIGEM - Telethon Institute of Genetics and Medicine
Naples, Italy

RobinAli
Institute of Ophthalmology - University College London
Division of Molecular Therapy Bath St
London, UK

SegoleneAyme
Orphanet - INSERM SC11
Hôpital Broussais
Paris, France

FatimaBosch
Universitat Autònoma de Barcelona
Centre de Biotecnologia Animal i Teràpia (CBATEG)
Bellaterra, Spain

JanBubenik
Academy of Sciences of the Czech Republic
Institute of Molecular Genetics
Prague, Czech Republic

ManuelCarrondo-PedroCruz
Instituto de Biologia e Tecnologia (IBET)
Oeiras, Portugal

ChristopherBaum
Hannover Medical School
Department of Experimental Haematology
Hannover, Germany

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gENE THERAPY

CLINIgENE

KlausCichutek
Paul-Ehrlich-Institut
Langen, Germany

NicoleDeglon
CEA - MIRCen
Orsay, France

GeorgeDickson
Royal Holloway & Bedford New College
School of Biological Science
Egham (Surrey), UK

GöstaGahrton
Karolinska Institutet
Department of Medicine, Huddinge University Hospital
Stockholm, Sweden

BerndGänsbacher
Klinikum rechts der Isar der Technischen Universität 
München
Institut für Experimentelle Onkologie und Therapiefor-
schung
Munich Germany

NicolasMermod
Université de Lausanne
Institut de Biotechnologie 
Lausanne, Switzerland

LluisMir
CNRS - UMR 8121
Institut Gustave-Roussy
Villejuif, France

PhilippeMoullier
CHU Hôtel Dieu Nantes
Laboratoire Thérapie Génique
Nantes, France

AmosPanet
The Hebrew University
Hadassah Medical School
Jerusalem, Israel

MarcPeschanski
ISTEM (Unité INSERM)
Evry Cedex, France

HansjörgHauser
Helmoltz Centre for Infection Research 
Braunschweig, Germany

Maria-GraziaRoncarolo
FCSR-TIGET
San Raffaele Telethon Institute for Gene Therapy
Milan, Italy

AndreasJacobs
Klinik für Neurologie der Universität zu Köln
Labors für Gentherapie & Molekulares Imaging am MPI 
für Neurologische Forschung
Cologne, Germany

DavidKlatzmann
UMR 7087-UPMC/CNRS-CERVI
Hôpital de la Pitié Salpêtrière
Paris, France

StefanKochanek
University of Ulm
Division of Gene Therapy
Ulm, Germany

DanielScherman
INSERM 
Paris, France

RichardVile
Mayo Clinic - Gene Therapy Programme
Rochester (Minnesota), USA

ChristofVonKalle
German Cancer Research Centre (DKFZ)
National Centre for Tumour Diseases (NCT) and 
Department of Translational Oncology, 
Heidelberg, Germany 

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MartinWisher
Bioreliance
Scotland, UK

VincentZuliani
Genosafe
Evry, France

LuçaySautron
Science Pratique SA 
Cachan, France

SeppoYla-Herttuala
University of Kuopio
A.I.Virtanen Institute
Kuopio, Finland

HeinzZwierzina
Innsbruck University
Department of Internal Medicine
Innsbruck, Austria

Anne-MarieMasquelier
Généthon
Evry, France

GillesAvenard
BioAlliance Pharma SA
Paris, France

CharlotteDalba
Epixis 
Paris, France

FredericHenry
Clean Cells
Bouffere, France

CharlesIrving
Theravir
Jerusalem, Israel

MonicaLuskyandJean-YvesBonnefoy
Transgene 
Strasbourg, France

KyriMitrophanous
Oxford Biomedica
Oxford, UK

FeliciaM.Rosenthal
CellGenix GmbH
Freiburg, Germany

MartinSchleef
Plasmid Factory
Bielefeld, Germany

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CONSERT

ConcertedSafetyandEfficiencyEvaluationofRetroviralTransgenesisforGene
TherapyofInheritedDiseases

gENE THERAPY






ContractNo 
Projecttype

LSHB-CT-2004-00242
IntegratedProject

ECcontribution  e 1163000

Startingdate 1November2004

Duration 48months

Website  www.gene-therapy.eu

Background and objectives:

The CONSERT project integrates leading Europe-
an activities for a structured implementation of 
novel therapies, using genetically modified post-
natal stem cells, with a focus on the treatment of 
immunodeficiencies,  haemoglob-
monogenic 
inopathies,  anaemias  and  storage  disorders. 
CONSERT  develops  and  evaluates  methods  for 
genetic  stem  cell  modification  with  wide  impli-
cations for many other disorders, including viral 
infection and cancer. 

A  central  theme  of  the  project  is  an  unbiased 
safety and efficiency evaluation of the key tech-
nology used in the genetic modification of rep-
licating somatic cells: retroviral vector-mediated 
transgenesis. This is made possible only through 
concerted  multi-centre  studies.  Lenti-,  spuma- 
and  gamma-retroviral  vectors  are  tailor-made 
for target disorders, and tested for potency and 
safety  in  preclinical  disease  models.  Designed 
with a translational aim, basic studies in stem cell 
biology and selectable marker technology com-
plement this research. 

A  highly  important  aspect  of  CONSERT  is  the 
molecular  and  clinical  monitoring  of  currently 
active  and  successful  clinical  trials  of  stem  cell 
gene  therapy  in  inherited  diseases. This  creates 
a  paradigmatic  data-mining  activity  to  obtain 
insights  into  crucial  issues  of  clonal  kinetics  of 
gene-modified cells in vivo. Molecular studies in 
precise cell systems and animal models provide 

the  mechanistic  understanding  of  transgene-
host interactions. The project generates the basis 
for  technology  development  and  promotes  pa-
tient safety.

Translational  dissemination  of  know-how  from 
academia  to  industry  creates  a  network  of  cell 
processing  manufacturers  with  large  economic 
potential,  and  prepares  researchers  for  future 
clinical studies with improved predictability. 

Genetic enhancement of cellular therapies is ex-
pected to have a major impact on the treatment 
of  numerous  inherited  and  acquired  disorders. 
Introducing  defined  genes  into  transplantable 
cells  will  ultimately  result  in  a  better  control  of 
their  expansion,  migration,  differentiation  and 
elimination in vivo, according to the individual’s 
unique  medical  condition.  Also,  novel  effector-
functions  can  be  introduced,  such  as  the  resto-
ration  of  defective  genes  or  protection  against 
drug toxicity, side effects or virus infection. Les-
sons  learnt  in  the  context  of  selected  inherited 
disorders  will  have  immediate  intellectual  and 
practical  consequences  for  the  development  of 
novel  treatment  options  against  acquired  dis-
eases such as cancer or viral infection.

European scientists and SMEs that hold key pat-
ents in gene transfer technologies and diagnos-
tic assays for patient monitoring have pioneered 
this field. All are members of CONSERT, underlin-
ing its high profile; they have also provided the 
first clinical evidence of a cure for inherited dis-

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gENE THERAPY

orders  of  haematopoiesis  by  cor-
rective  gene  transfer  into  haemat-
opoietic stem/progenitor cells.

Stem cell Biology and Transduction
In vivo Selection approaches
Safety

Fundamental
Knowledge

Given  this  background,  CONSERT’s 
major  objective  is  the  concerted 
safety  and  efficiency  evaluation  of 
retroviral  transgenesis.  This  is  ex-
emplified  with  selected  inherited 
disorders  as  clinical  targets,  which 
are in urgent need of the develop-
ment of novel curative approaches. 
CONSERT’s  work  programme  re-
flects that the full potential of gene 
therapy  can  only  be  developed 
through an integrated approach that targets ex-
isting biological, clinical, technological, commer-
cial and ethical hurdles. 

CONSERT’s  scientific  structure  of  integrating 
work  packages  (WPs)  directed  at  fundamental 
knowledge  to  treat  specific  diseases  with  cura-
tive intent, as well as with a strong genomics and 
manufacturing component, and a proactive dis-

Disease Specific
Projects

Red cell disorders
Wiskott-Aldrich Syndrome
Leukodystrophies & Lysosomal storage disorders
Chronic Granulomatous Disease

Manufacturing

Interface
projects

A Genomic Interface Program

Outreach &
Training

Ethics
Regulatory, IPR and legal issues
A tailored Education and Exchange programme

seminations and ethics component, can be visu-
alised, in a nutshell, as demonstrated below: 

 Main findings:

Highlights of the work completed are presented 
below.

Schematic representation of gene therapy for inherited 
diseases. Areas shaded in blue, mark pharmaceutical 
involvement and potential.

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gENE THERAPY

CONSERT

Members  of  CONSERT  have  developed  break-
throughs  in  basic  vector  technology,  molecular 
analysis of gene insertion profiles, and upscaling 
of cell manipulation procedures for clinical use. 

As  with  any  new  therapeutic  approach,  gene 
transfer may also induce novel kinds of side ef-
fects. Somewhat reflecting the leading status of 
the project’s research and application, members 
of  CONSERT  have  also  demonstrated  the  first 
cases of serious adverse events of gene transfer 
into haematopoietic cells, and designed preclini-
cal as well as clinical approaches to address and 
prevent the underlying mechanisms. 

The population of stem cells that allows for the 
complete  restoration  of  lymphohaematopoiesis 
is difficult to enrich and maintain in vitro.  Several 
participants have made a breakthrough by dem-
onstrating  that  growth  factor  stimulated  stem 
cells ex vivo under serum free conditions do not 
lose their repopulating capacity and actually can 
be  amplified  ex  vivo. This  is  now  further  imple-
mented in preclinical evaluation protocols. State-
of-the-art  preclinical  assay  systems  have  been 
made available to the project, which include pri-
mary cell culture models, murine models of regu-
lar  and  genetically  altered  haematopoiesis,  and 
immunodeficient mice for the preclinical study of 
human haematopoiesis, to be validated by stud-
ies  in  non-human  primates  if  ethically  justified. 
These are needed to address how the quality of 
cell purification, novel culture conditions, vector 
improvements and cell application protocols can 
be improved, to achieve long-term polyclonal re-
constitution of haematopoiesis in vivo.

Leukaemia  induction  is  still  a  rare  complication 
of  stem  cell  gene  therapy,  which  requires  the 
simultaneous  existence  of  clonal  dominance 
(which  might  even  contribute  to  the  therapeu-
tic effect of retroviral gene transfer) of otherwise 
benign  haematopoietic  cells  and  additional 
cancerogenic signal alterations. Importantly, the 
partners  have  successfully  established  various 

mouse  models  in  which  the  impact  of  vector 
design on insertional mutagenesis can be evalu-
ated directly. 

Ever-increasing data obtained by CONSERT teams 
suggest that the risk of insertional mutagenesis 
can be strongly reduced by improving the vector 
design  (using  lentiviral  vectors  and  other  types 
of integrating vectors with redesigned transgene 
cassettes),  highly  purified  and  limited  numbers 
of cells used, and the use of cell lineage specific 
expression  of  the  transgenes.  The  consortium 
has succeeded in developing a new generation 
of self-inactivating gammaretroviral and lentivi-
ral  vectors,  that  are  currently  tested  for  efficacy 
and safety.

In  the  safety  evaluation  and  genomics  work 
packages, the consortium has unravelled the re-
lationship between gammaretroviral integration 
and gene expression profiles of immature stem 
cells.  The  results  have  provided  novel  insights 
into  retroviral  transgenesis  and  the  effects  of 
retroviral integrations on stem cell behaviour, as 
well as yielded novel insights into stem cell biol-
ogy. These results provide a solid basis for safety 
evaluation  of  the  new  generation  of  vectors 
constructed.

Elaborating  from  our  paradigms  of  successful 
clinical studies and side effect mana¬g¬¬e¬ment, 
the project is addressing a number of inherited 
disorders  for  which  a  sufficient  conventional 
therapy is not available besides allogeneic bone 
marrow  transplantation  (alloBMT).  Several  of 
the  diseases  chosen  are  paradigms  for  a  selec-
tive advantage of gene-modified cells (SCID-X1, 
ADA-SCID,  Wiskott-Aldrich-Syndrome,  Fanconi 
anemia),  while  others  (chronic  granulomatous 
disease, thalassemia, Diamond-Blackfan anemia) 
require  stronger  preparation  of  the  patient  for 
cell transplantation or the co-expression of a lin-
eage-restricted therapeutic gene with a selecta-
ble gene operative at the level of stem cells. 

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gENE THERAPY

Animal models are available for all disorders, al-
lowing  disease-specific  preclinical  potency  and 
safety  evaluation.  Among  the  clinical  targets 
chosen,  thalassemia  ultimately  has  the  greatest 
social and healthcare impact. The metabolic stor-
age  disorder  metachromatic  leukodystrophy 
and  Wiskott-Aldrich-Syndrome,  have  reached 
the stage of pharmaceutical development pre-
paring for clinical trial. Members of the consor-
tium actively conduct clinical trials for SCID-X1 
and ADA-SCID. 

In  future  commercialisation  development,  the 
leading role of European science in gene therapy 
development for inherited diseases as represent-
ed in CONSERT, also translates into SME activities 
with  significant  potential  for  intellectual  prop-
erty  management  and  product  development. 
Three SMEs or non-profit organisations engaged 
with CONSERT have documented their expertise 
in  retroviral  technology  upscaling,  clinical  ap-
plication of advanced cell products and process 
standardisation. Moreover, insertion site invento-
ries  arising  from  current  preclinical  and  clinical 
data mining approaches of retroviral transgene-
sis, create the basis for further SME participations, 
in which key technologies of functional genom-
ics are developed and implemented. The IPR po-
sition of the consortium has been fully evaluated, 
and alternative models for exploitation of the re-
sults are under development.

Public interactions, ethical reflections and teach-
ing  activities  are  integral  components  of  CON-
SERT’s  scientific  and  technological  objectives. 
Expanding the well-documented activities of in-
dividual members with national and EU regula-
tory agencies (including EMEA), patient interest 
groups, ethical committees and gene therapy so-
cieties (including ESGT), CONSERT is implement-
ing a central ethics project to address potential 
conflicts  arising  from  cutting-edge  technology 
developments in somatic transgenesis. 

The  annual  scientific  meetings  are  designed  to 

be  a  major  educational  event  for  the  post-docs 
and PhD students working within the project. In 
those  meetings,  the  ethical  reflection  has  been 
implemented as a plenary workshop for all partic-
ipants. Two ethics conferences, directed at estab-
lishing a European consensus for the ethical con-
siderations involved in the development of gene 
therapy for inherited diseases, are scheduled for 
2007. In addition, the consortium participates in 
European training courses in collaboration with 
other  European  projects  and  patient  organisa-
tions, and periodically approaches the lay press. 
The  European  umbrella  patient  organisation 
EGAN has been included as a partner in the sec-
ond phase of the project, to lead the patient and 
public  information  platform  of  the  consortium. 
The consortium is maintaining a website for ac-
cess to public and professional information.

Major publications

Berglin-Enquist, I., Nilsson, E., Ooka, A., Månsson, 
J.E., Olsson, K., Ehinger, M., Brady, R.O., Richter, J., 
Karlsson, S., ‘Effective cell and gene therapy in a 
murine model of Gaucher disease’, Proc. Natl. Acad. 
Sci. USA, 2006, 103:13819-13824. E-publ Sept 5.

Fuchs,  M., ‘Gene  therapy.  An  ethical  profile  of  a 
new medical territory,’ Journal of Gene Medicine, 
2006, 8(11): p. 1358-1362.

Kustikova, O., Fehse, B., Modlich, U., Düllmann, J., 
Kamino, K., von Neuhoff, N., Schlegelberger, B., Li, 
Z.,  Baum,  C., ‘Clonal  dominance  of  haematopoi-
etic stem cells triggered by retroviral gene mark-
ing’, Science, 2005, 308: 1171-1174

Kustikova,  O.,  Geiger,  H.,  Li,  Z.,  Brugman,  M.H., 
Chambers,  S.M.,  Shaw,  C.A.,  Pike-Overzet,  K.,  de 
Ridder, D., Staal, F.J.T., Keudell, G., Cornils, K., Nat-
tamai,  K.J.,  Modlich,  U.,  Wagemaker,  G.,  Goodell, 
M.A.,  Fehse,  B.,  Baum,  C., ‘Retroviral  vector  inser-
tion  sites  associated  with  dominant  haemat-
opoietic clones mark “stemness” pathways’, Blood, 
2006, E-publ. Nov 21.

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gENE THERAPY

CONSERT

Montini,  E.,  Cesana,  D.,  Schmidt,  M.,  Sanvito,  F., 
Ponzoni, M., Bartholomae, C., Sergi Sergi, L., Ben-
edicenti,  F.,  Ambrosi,  A.,  Di  Serio,  C.,  Doglioni,  C., 
von Kalle, C., Naldini, L., ‘Haematopoietic stem cell 
gene transfer in a tumor-prone mouse model un-
covers low genotoxicity of lentiviral vector inte-
gration’, Nature Biotechnol, 2006, (6):687-96.

Ott, M.G., Schmidt, M., Schwarzwaelder, K., Stein, 
S., Siler, U., Koehl, U., Glimm, H., Kuhlcke, K., Schilz, 
A., Kunkel, H., Naundorf, S., Brinkmann, A., Deich-
mann, A., Fischer, M., Ball, C., Pilz, I., Dunbar, C., ,Du 
Y., Jenkins, N.A., Copeland, N.G., Luthi, U., Hassan, 
M., Thrasher, A.J., Hoelzer, D., von Kalle, C., Seger, R., 
Grez, M., ‘Correction of X-linked chronic granulo-
matous disease by gene therapy, augmented by 
insertional  activation  of  MDS1-EVI1,  PRDM16  or 
SETBP1’,  Nature Medicine, 2006, Apr;12(4):401-9. 

Pike-Overzet, K., de Ridder, D., Weerkamp, F., Baert, 
M.R., Verstegen, M.M., Brugman, M.H., Howe, S.J., 
Reinders,  M.J., Thrasher,  A.J., Wagemaker,  G.,  van 
Dongen, J.J., Staal, F.J., ‘Gene therapy: is IL2RG on-
cogenic in T-cell development?’ Nature, 2006, Sep 
21;443(7109):E5; discussion E6-7. 

Thrasher, A.J., Gaspar, H.B., Baum, C., Modlich, U., 
Schambach,  A.,  Candotti,  F.,  Otsu,  M.,  Sorrentino, 
B., Scobie, L., Cameron, E., Blyth, K., Neil, J., Abina, 
S.H., Cavazzana-Calvo, M., Fischer, A., ‘Gene thera-
py:  X-SCID  transgene  leukaemogenicity’,  Nature, 
2006, Sep 21;443(7109):E5-6; discussion E6-7.

Verhoeyen, E., Wiznerowicz, M., Olivier, D., Izac, B., 
Trono,  D.,  Dubart-Kupperschmitt,  A.,  Cosset,  F.L., 
‘Novel  lentiviral  vectors  displaying  “early  act-
ing  cytokines”  selectively  promote  survival  and 
transduction of NOD/SCID repopulating human 
haematopoietic  stem  cells’,    Blood,  2005,  Nov 
15;106(10):3386-95. 

Yanez-Munoz,  R.J.,  Balaggan,  K.S.,  MacNeil,  A., 
Howe,  S.J.,  Schmidt,  M.,  Smith,  A.J.,  Buch,  P.,  Ma-
cLaren, R.E., Anderson, P.N., Barker, S.E., Duran, Y., 
Bartholomae,  C.,  von  Kalle,  C.,  Heckenlively,  J.R., 

Kinnon,  C.,  Ali,  R.R., Thrasher,  A.J., ‘Effective  gene 
therapy  with  nonintegrating  lentiviral  vectors’, 
Nature Medicine, 2006, Mar;12(3):348-53. 

Coordinator 

GerardWagemaker
Erasmus University Medical Centre
Faculty Building, Department of Haematology, Ee 1314
Dr. Molewaterplein 50
3015 GE Rotterdam, Netherlands
PO Box 2040, 3000 CA Rotterdam, Netherlands
E-mail: g.wagemaker@erasmusmc.nl

Partners

DidierTrono
École Polytechnique Fédérale de Lausanne 
Laboratory of Virology and Genetics 
Lausanne, Switzerland

ChristopherBaum
Hannover Medical School
Department of Haematology Haemostaseology and 
Oncology
Laboratory of Experimental Cell Therapy 
Hannover, Germany

ChristofVonKalle
National Centre for Tumour Diseases (NCT) 
Heidelberg, Germany

KlausKuehlcke
European Institute for Research and Development of 
Transplantation Studies AG 
Oberstein, Germany

MichaelFuchs
Institut für Wissenschaft und Ethik
Section of Biomedical Ethics
Bonn, Germany

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gENE THERAPY

ThomasM.Pohl
GATC Biotech AG
Konstanz, Germany 

LuigiNaldini
Fondazione Centro San Raffaele del Monte Tabor 
DIBIT – TIGET
Milan, Italy

JordiBarquinero
Centre de Transfució i Banc de Teixit 
Unitat de Diagnostic i Terapia Molecular 
Barcelona, Spain

JuanA.Bueren
Centro de Investigaciones Energeticas Mediambientales 
Y Technolgicas
Haematopoiesis Programme
Madrid, Spain

François-LoïcCosset
INSERM Unit 412 
Laboratoire de Vectorologie Retrovirale et 
Therapie Genique –ENS 
Lyon, France

MariaGraziaRoncarolo
Università Vita-Salute San Raffaele 
Faculty of Medicine
Milan, Italy

LouisevandenBos
Science & Technology Transfer 
Rotterdam, Netherlands

StefanKarlsson
Lunds Universitet 
Laboratory of Medicine
Medical Faculty
Moleculair Medicine and Gene Therapy
Lund, Sweden

AlainFischerandMarinaCavazzana-Calvo
INSERM Unit 429 
 Hopital Necker Enfants Malades
Paris, France

AdrianThrasher
University College London
Molecular Immunology Unit, Institute of Child Health
London, UK

AnneGaly
Généthon
Evry, France

WilliamSaurin
Genomining
Montrouge, France

NicholasAnagnou
National and Kapodistrian University of Athens 
Basic Sciences, University of Athens School of Medicine, 
Laboratory of Biology
Athens, Greece

FulvioMavilio
Molecular Medicine S.p.A
Discovery
Milan, Italy

MaryCollins
University College London 
Immunology and Molecular Pathology
London, UK

GeorgeVassilopoulos
Foundation of Medical and Biological Research of the 
Academy of Athens 
Laboratory of Cell and Gene Therapy, Centre of Basic 
Research
Athens, Greece

ManuelGrez
Georg-Speyer Haus
Frankfurt, Germany

CorOosterwijk
European Genetic Alliances Network
Soestdijk, Netherlands

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gIANT

Genetherapy:anintegratedapproach

gENE THERAPY






ContractNo 
Projecttype

LSHB-CT-2004-12087
IntegratedProject

ECcontribution  e 9700000

Startingdate 1January200
Duration 60months

Website  www.giant.eu.com

Background and objectives:

Screening  for  prostate  cancer  is  likely  to  have 
an impact on the spectrum of disease, changing 
the distribution towards organ-confined disease 
that is susceptible to radical surgery, radiothera-
py,  and  in  80%  of  cases,  anti-androgen  therapy. 
However, even with such improvements and ad-
vancements, the disease burden in Europe in an 
ageing male population will be substantial, con-
sidering  the  expense  involved  in  screening  as 
well as in radical surgery (with impotence as the 
most  common  consequence  and  incontinence 
encountered in 3-5% of cases). 

In the longer term, when the tumour has escaped 
the prostatic capsule, and anti-androgen therapy 
has  failed,  the  tumour  returns  and  is  almost  in-
variably fatal within 18-24 months. At this stage, 
most  conventional  cytotoxic  therapies  are  inef-
fective, although immunotherapeutic approach-
es  and  taxotere  treatment  have  shown  some 
promise. Even these improvements result in little 
more than a two-month extension of life.

A novel gene-based therapy, which can take ac-
count of the heterogeneity of the disease, will of-
fer the possibility of extended life of good quality, 
even if all of the tumour cells are not destroyed. 
To be a successful vehicle for gene therapy, each 
vector type (both viral or non-viral) has its own 
particular qualities and limitations, and its degree 
of  development  also  depends  on  the  intended 
medical  application.  The  choice  of  the  target, 

the  transgene,  and  duration  and  stability  of  its 
expression  all  influence  final  vector  choice  and 
design. With ongoing research and discoveries in 
the field of molecular genetics, it is expected that 
new generations of genetic vectors will be essen-
tial for the translation of molecular research into 
clinical practice.

EGFP expressing virus Infected xenografts
 Adenovirus CMV EGFP

Approach and methodology:

The  GIANT  project  will  provide  an  international 
resource to apply innovative technologies for the 
modification of existing gene therapy vectors, fo-
cusing  on  increased  prostate  targeting  and  de-

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gENE THERAPY

creased  vector  immunogenicity  by  ‘stealthing’. 
The  pre-existing  and  innate  immune  response 
against viruses requires the inoculation of large 
amounts of virus to produce a therapeutic effect 
with a high cost per treatment. The GIANT modi-
fied vectors will be tested in uniform in vitro and 
in vivo  models  of  human  prostate  for  improved 
efficacy, prior to GIANT sponsored and organised 
Phase I trials. 

However, this translational action will also serve 
as a template for the development of innovative 
treatments based on gene therapy, which can be 
applied in generic terms to other tumour types, 
using  appropriate,  tumour-specific  targeting. 
The project also seeks to harmonise the regula-
tions  for  clinical  trials  in  Europe,  to  encourage 
multicentre, multinational clinical trials of cancer 
gene therapy using common stocks of GLP grade 
viruses as a ‘gene medicine’ in the same way as 
small molecules can now be tested in the clinic.

Expected outcome:

The  GIANT  consortium  seeks  to  secure  the 
following:

•  generation  of  vectors  which  prefer-
entially  attach  to  and  penetrate  prostate 
tumour cells;
•  preferential  expression  of  optimised 
therapeutic  genes  in  prostate  tumour 
cells,  relative  to  other  cell  and  tumour 
types;
• 
combination of the above to eliminate 
the expression of the prototype therapeu-
tic genes (HSVtk), and growth of oncolytic 
adenoviruses in adjacent tissues;
•  optimisation of therapeutic gene/pro-
drug  combinations  to  maximise  thera-
peutic effects;
• 
further  development  of  advanced 
non-viral  vector  systems,  and  assessment 
of  their  activity  preclinically  in  controlled 
comparisons  with  the  currently  accepted 
gold  standard  vector  system  for  cancer 

gene therapy (human adenovirus vectors);
•  prevention of attachment of retarget-
ed  vectors  to  cells  and  matrix  elements 
within blood vessels and other tissues and 
organs, to permit ultimate intravenous ap-
plication  of  gene  therapy,  and  minimise 
treatment side effects;
•  promotion of gene therapy as a valid 
anti-cancer  strategy  by  early  design  and 
conduction of a Phase I trial, using the first 
generation of viral vectors.

Main findings:

The project partners have significantly improved 
the  technology  to  incorporate  polypeptide-lig-
ands in the HI loop of the adenovirus fibre. Fur-
thermore,  adenovirus  fibres  have  been  entirely 
replaced by a single-chain TCR and the viral pro-
tein  IX  molecules  have  been  fused  with  scTCRs, 
with  affibodies,  as  well  as  with  scFv  fragments. 
This  technology  allows  the  modified  viruses  to 
bind  with  enhanced  affinity  to  new  target  cells 
(e.g.prostate cancers) and to target the delivery 
of therapeutic genes.
New  molecules  have  been  designed  using  ad-
vanced  polymer  chemistry,  to  de-  and  re-target 
adenoviruses  by  coating  the  viral  particles. The 
effectiveness  of  these  novel  polymer  coated 
vectors has been determined in vivo. A novel hu-
man  phage  display  library  has  been  generated 
to  provide  new  anti-PSMA  scFv,  Affibodies  and 
MHC-restricted  Fab  fragments  directed  against 
the  Prostate-Specific  Membrane  Antigen.  Good 
manufacturing  practice  grade  polyethylene-
imine has been generated to initiate clinical trials 
of non-viral vectors. 

GIANT has also achieved the retargeting of non-
viral vectors with cell-specific receptors and hy-
drolytic  degradation/reduction.  Efficacy  of  the 
Ad[I/PPT-E1A] prostate-specific oncolytic adeno-
virus has been determined in vitro and in vivo as 
a  prelude  to  the  first  GIANT  clinical  trial.  Finally, 
logistics  for  a  three-country  phase  I  trial  of  the 

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gENE THERAPY

gIANT

Ad[I/PPT-E1A] prostate-specific oncolytic adeno-
virus are mostly complete, and clinical grade ma-
terial has been ordered for the trial.

Major publications

Kreppel, F., Gackowski, J., Schmidt, E., Kochanek, S., 
‘Combined  Genetic  and  Chemical  Capsid  Modi-
fications  Enable  Flexible  and  Efficient  De-  and 
Retargeting  of  Adenovirus  Vectors’,  Molecular 
therapy:  The  Journal  of  the  American  Society  of 
Gene Therapy, 2005, July: 12(1): 107-17

Kraaij, R., van Rijswijk, P., Oomen, M.H.A., Haisma, 
H.J.,  Bangma,  C.H., ‘Prostate  Specific  Membrane 
Antigen (PSMA) Is a Tissue-Specific Target for Ad-
enoviral Transduction of Prostate Cancer In vitro’, 
The Prostate, 2005, February 15; 62(3): 253-9 

Vellinga, J., De Vrij, J., Myhre, S., Uil, T., Martineau, 
P., Lindholm, L., Hoeben, R.C., ‘Efficient Incorpora-
tion  of  a  Functional  Hyper-Stable  Single-Chain 
Antibody Fragment Protein-IX Fusion in the Ad-
enovirus Capsid’, Gene Ther, 2007, Apr;14(8):664-
70. Epub 2007 Feb 1. 

Dissemination
‘New  treatments  for  an  old  man’s  disease’  The 
House, Maitland, N.J., 2007, 32: 36-37.

Patents

100

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gENE THERAPY

LeifLindholm
Got-a-Gene AB
Kullavik, Sweden

WystkevanWeerden
Scuron
Rotterdam, Netherlands

NormanMaitland
Procure
York, UK

ErnstWagner
Ludwig-Maximilians-Universitat
Munich, Germany

Jean-PaulBehr
Université Louis Pasteur de Strasbourg
Illkirch, France

PatrickErbacher
Polyplus Transfection
Illkirch, France

KerryFisher
Hybrid Systems
Oxford, UK

Coordinator

NormanJ.Maitland
University of York 
YCR Cancer Research Unit
Department of Biology
York YO10 5YW, UK
E-mail: njm9@york.ac.uk

Scientific coordinator 

EllenSchenk-Braat
Erasmus MC University Medical Centre
Department of Urology H1072 
Dr Molewaterplein 40 
3015 GD Rotterdam, Netherlands 
E-mail: e.schenk-braat@erasmusmc.nl 

Partners

ChrisBangma
Erasmus MC University Medical Centre
Rotterdam, Netherlands.

MagnusEssand
Uppsala University
Uppsala, Sweden

LenSeymour
University of Oxford
Oxford, UK

RobHoeben
Leiden University Medical Centre
Leiden, Netherlands

StefanKochanek
University of Ulm
Ulm, Germany

KarelUlbrich
Academy of Sciences of the Czech Republic
Prague, Czech Republic

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101

BACULOgENES

Useofbaculovirusasavectorforgenetherapy

gENE THERAPY

ContractNo 
Projecttype

LHSB-CT-2006-03741
SpecificTargetedResearchProject

ECcontribution  e 2499746

Startingdate 1January2007
Duration 36months






Background and objectives:

Gene therapy is a technique that delivers thera-
peutic  nucleic  acids  into  somatic  cells;  it  is  one 
of  the  most  promising  therapeutic  methods 
under  development  for  treating  a  large  scope 
of  pathologies,  ranging  from  genetic  disorders 
(e.g.  myopathies)  to  degeneration  syndromes 
or  cancers.  The  potential  of  gene  therapy  has 
not  yet  been  fully  exploited,  mainly  because  of 
significant limitations related to the safety, gene 
delivery  capacities  and  several  other  properties 
of the currently used vectors. 

Baculoviruses (BVs) are insect pathogenic DNA vi-
ruses and are not known to replicate in mamma-
lian cells; this gives them an advantage in terms 
of  safety  over  classical  mammalian  viruses  cur-
rently used as vectors, like the adeno-associated 
virus  (AAV),  the  adenovirus,  murine  retroviruses 
and lentiviruses. The most promising baculovirus 
for gene therapy is the well-known Autographa 
californica  multiple  nucleopolyhedrovirus  (Ac-
MNPV). It is inherently safe and can deliver large 
pieces of DNA on its genome (≥50 Kbp). 

BV replication and virus production does not occur 
in mammalian cells and BV is not known to be asso-
ciated with any human disease. However, by using 
a vertebrate active expression cassette as a part of 
baculovirus genome, efficient gene expression can 
also be directed in non-target cells. A large range of 
vertebrate cells has been shown to be permissive 
for AcMNPV transduction, both in vitro and in vivo. 

BV technology has been used for years for pro-
ducing  recombinant  proteins,  and  thus  large-
scale production technology is readily adaptable 
for the exploitation of gene therapy approaches. 
In addition, BV vectors can be used efficiently for 
producing  other  gene  therapy  vectors  such  as 
AAVs. The BV genome is well-known, and several 
selective  targeting  approaches  engineered  into 
the virus envelope and capsid have been devel-
oped. 

BACULOGENES  aims  to  develop  clinically  suit-
able methods for the development, production, 
testing  and  validation  of  next-generation  stabi-
lised  and  selective  BV  vectors  for  gene  therapy 
applications,  as  well  as  to  optimise  the  produc-
tion of new AAV serotype vectors. Target diseases 
for in vivo gene delivery with selectively targeted 
BV include muscle disorders, age-related macular 
degeneration and prostate cancer. 

The BACULOGENES consortium consists of eight 
partners from six countries, including pioneers in 
the use of BVs for mammalian gene transfer appli-
cations, and two major established gene therapy 
vector-producing companies in the EU. The con-
sortium will devote its efforts not only to BV gene 
therapy applications, but also to the development 
of  large-scale  production,  downstream  process-
ing, purification and analysis methods. The quality 
control and validation assays, and all issues relat-
ed to regulatory aspects required for the clinical 
exploitation of BV technology, will be covered.

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gENE THERAPY

Approach and methodology:

Coordinator 

The innovative strategy of BACULOGENES relies 
on the original engineering of BVs (AcMNPV) to 
make them safe, specific and efficient vectors for 
gene therapy. BACULOGENES’ approach consists 
of  stabilising  the  BV  genome  through  deletion 
and  insertion  of  specific  sequences,  in  combi-
nation  with  transgenes  relevant  to  the  disease 
targeted.  The ‘stabilised’  BVs  will  then  be  engi-
neered as follows: (a) to enhance the capacity to 
deliver the therapeutic gene(s) to the right cells, 
(b) to optimise the therapeutic gene expression 
in the right cells, and (c) to make BVs less immu-
nogenic and potentially invisible for the immune 
system (stealth virus). Such improvements will be 
obtained  through  envelope  protein,  capsid  and 
genome modifications. 

In  parallel,  the  baculovirus  expression  system 
will be optimised for the production of different 
serotypes of AAV by using stabilised constructs. 
In addition, the development of baculovirus con-
structs allowing the production of recombinant 
AAV without the concomitant production of bac-
ulovirus is planned. 

Expected outcome:

BACULOGENES  will  strengthen  European  lead-
ership,  knowledge  and  competitiveness  in  BV 
technology,  through  the  delivery  of  novel  and 
validated efficient vectors, as well as in platform 
technology, for the exploitation of potential clini-
cal applications of gene therapy. This project will 
thus pave the way for BVs to be used in clinical 
applications,  an  area  not  yet  explored  with  ex-
periments,  and  will  provide  an  optimised  BV-
based AAV platform for gene therapy purposes. 
The project will also offer the biotech communi-
ty a highly efficient manufacturing process, and 
associated QC methods for BVs processing.

JohnMartin
Ark Therapeutics Ltd 
79 New Cavendish Street 
London W1W 6XB, UK
E-mail: john.martin@arktherapeutics.com  

Scientific coordinator 

AnssiMähönen
Ark Therapeutics Oy
Kuopio, Finland
E-mail: anssi.mahonen@arktherapeutics.com 

Partners

NickHunt
Altonabiotec
Hamburg, Germany

PaulaAlves
Instituto de Biologia Experimental e Tecnologica (IBET)
Oeiras, Portugal

MoniqueM.vanOers
Wageningen University 
Laboratory of Virology 
Wageningen, Netherlands 

KariAirenne
University of Kuopio
Kuopio, Finland 

LindsayGeorgopoulos
University of York
YCR Cancer Research Unit
York, UK

Otto-WilhelmMerten
Généthon
Evry Cedex, France

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103

THOvLEN

Targeted herpesvirus-derived oncolytic vectors for liver cancer European
network

gENE THERAPY






ContractNo 
Projecttype

LSHB-CT-200-018649
SpecificTargetedResearchProject

ECcontribution  e 2494460

Startingdate 1January2006
Duration 36months

Website  http://www.thovlen.eu

Background and objectives:

THOVLEN  seeks  to  develop  safe  and  efficient 
herpes simplex virus type 1 (HSV-1)-derived on-
colytic  vectors,  designed  to  strictly  target  and 
eradicate  human  hepatocellular  carcinomas 
(HCCs), the most common liver cancer in adults. 
HSV-1 is certainly one of the most promising vi-
ral  platforms  for  the  development  of  improved 
oncolytic  vectors,  as  anticipated  by  the  unique 
biological properties of this virus and confirmed 
by the encouraging results generated by clinical 
trials in gliomas. However, the first generations of 
oncolytic HSV-1 vectors have also shown limita-
tions regarding efficacy and safety. 

New  generations  of  innovative  HSV-1  vectors, 
with improved potency and safety, are required 
before  the  oncolytic  strategy  using  HSV-1  be-
comes a standard therapeutic reality in the fight 
against cancer. This is the goal of THOVLEN. One 
of  the  most  important  innovative  contributions 
of this project concerns the overall approach to-
wards the advancement of HSV-1-based oncolytic 
viruses. Instead of focusing on the development 
of  vectors  carrying  deletions,  and  in  particular 
virus  genes, THOVLEN  will  engineer  competent, 
but  replication-restricted  HSV-1  vectors,  strictly 
targeted  to  HCC.  These  vectors  will  combine 
multiple HCC-targeting approaches, both at the 
level of entry and at the level of gene expression 
and replication, and will be able to multiply and 
spread only in HCC, while displaying no virulence 
in normal healthy tissues.

Another  novelty  is  related  to  the  ability  of  the 
HSV-1 vectors to permit a sophisticated and flex-
ible combined approach against HCC. That is, in 
addition  to  optimising  the  oncolytic  properties 
of HSV-1 vectors, THOVLEN will exploit the very 
large transgenic capability of HSV-1, to generate 
vectors that will simultaneously display multiple 
and  multimodal  anti-tumour  activities  acting 
either locally or systemically. These include com-
bined  expressions  of  anti-angiogenic,  immune-
modulatory, and oncolytic proteins.

Approach and methodology:

THOVLEN  will  design  HCC-targeted  virus  vec-
tors  that  will  simultaneously  display  multiple 
targeting  elements  acting  at  different  steps  of 
the virus life cycle, in order to ensure maximum 
aggressiveness  for  HCC  cells  with  minimum  or 
no  virulence  for  healthy  tissues.  Moreover,  the 
unique  advantage  of  the  HSV  genome  to  carry 
about 40 kbs of foreign DNA will be exploited in 
the context of designing a multimodal approach 
for cancer therapy, required for improvement of 
the inherent anti-tumour activity of the virus. 

The  availability  and  expertise  in  the  use  of  sev-
eral well-defined animal models for liver cancer, 
will allow the consortium to evaluate the safety 
and efficacy of their vectors in relevant systems. 
Through fundamental research, they will gener-
ate  novel  genomic  and  proteomic  information 
on  the  interactions  between  the  oncolytic  vec-
tors and the normal and cancer cells, which will 

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gENE THERAPY

The  first  of  these  recombinant  vectors  has  al-
ready  been  achieved  and THOVLEN  is  currently 
studying their biological properties. At the same 
time, animal models and immune tools that will 
allow assessments of the anti-oncogenic proper-
ties, the toxicity and the immunogenicity of the 
vectors, have been created. 

Lastly,  THOVLEN  is  now  conducting  proteomic 
and transcriptomal studies to compare infected 
versus non-infected hepatomas, in order to iden-
tify  changes  in  the  expression  patterns  of  the 
infected cells, as well as a way to identify novel 
activated promoters that could eventually allow 
improving the targeting of the oncolytic vectors. 
With several collaborations forged with hospitals 
in France and Greece, fresh human hepatocytes 
have been secured, helping to serve to evaluate 
the impact of the oncolytic vectors in these cells, 
and to confirm that the vectors can replicate and 
disseminate  in  cultured  hepatomas,  but  not  in 
cultured hepatocytes.

guide the rational design of vectors targeted to 
HCC  cells.  The  emerging  results  will  be  the  im-
provement of the vector oncolytic potency and 
of safety. Different ways of producing HSV-1 vec-
tors conceived to specifically penetrate, express 
genes, and replicate into HCC will be investigat-
ed, thus killing these cells and allowing the virus 
to spread and infect other tumour cells. 

These vectors should be unable to replicate and 
destroy  normal  surrounding  cells.  In  addition 
to  their  inherent  targeted  oncolytic  potential, 
these vectors will express enhancing transgenic 
sequences,  encoding  immune-modulators,  anti-
angiogenic  molecules,  fusion  proteins,  or  toxic 
proteins, which are expected to have an additive 
negative effect on tumour growth. 

Expected outcome:

Once  the  project  ends,  the  consortium  expects 
to  produce  a  number  of  HSV-1-based  oncolytic 
vectors specifically targeted to treat HCC. These 
vectors  will  combine  targeting  and  enhancing 
functions,  which  will  be  evaluated  for  efficacy 
and  toxicity  on  different  HCC  animal  models, 
including standard and transgenic mice, as well 
as woodchucks. The potential applications of the 
results and observations generated by THOVLEN 
concern  the  development  of  novel  strategies 
for  the  treatment  of  primary  liver  cancers  of 
humans.

Main findings and results:

In  the  first  year  of  the  project,  the  consortium 
succeeded in constructing the majority of tran-
scription units expressing essential HSV-1 genes 
under  the  control  of  cancer-specific  promoters. 
They  also  set  up  transcription  units  expressing 
HSV-1 glycoproteins that were modified to allow 
specific  entry  of  the  vectors  in  liver  cells. These 
genes  are  currently  being  introduced  into  the 
HSV-1  genome  so  as  to  generate  the  recom-
binant viruses. 

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New Therapies – Gene Therapy

10

gENE THERAPY

THOvLEN

Coordinator

AlbertoEpstein
Centre de Génétique Moléculaire et Cellulaire
Université Claude Bernard Lyon 1
Lyon, France 
E-mail: epstein@cgmc.univ-lyon1.fr

Partners

RobertoManservigi
Dipartimento di Medicina Sperimentale e Diagnostica
Sezione di Microbiologia
Università degli Studi di Ferrara
Ferrara, Italy

ThomasBrocker
Ludwig-Maximilians-Universitaet Muenchen
Institute for Immunology
Munich, Germany

PenelopeMavromara
Hellenic Pasteur Institute
Athens, Greece

RubenHernandezAlcoceba
Fundacion para la investigacion medica aplicada (FIMA)
Pamplona, Spain

FernandoCorrales
Fundacion para la investigacion medica aplicada (FIMA)
Pamplona, Spain

AndresCrespo
Genopoietic
Miribel, France

Jean-JacquesDiaz
Centre de Génétique Moléculaire et Cellulaire
CNRS – UMR 5534
Université Claude Bernard Lyon I (UCBL)
Villeurbanne, France

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gENE THERAPY

THERAdPOX

Optimisedandnoveloncolyticadenovirusesandpoxvirusesinthetreatment
ofcancer:virotherapycombinedwithmolecularchemotherapy






ContractNo 
Projecttype

LSHB-CT-200-018700
SpecificTargetedResearchProject

ECcontribution  e 2411006

Startingdate 1December200
Duration 36months

Website  www.theradpox.org

Background and objectives: 

Today, cancer is the second-leading cause of dis-
ease-induced  mortality  worldwide.  Among  the 
innovative  treatments  for  cancer,  virotherapy 
holds great promise. Oncolytic viruses (OVs) are 
replicating  micro-organisms  (viruses)  that  have 
been  selected  or  engineered,  so  as  to  grow  in-
side and kill tumour cells. It is well known that as 
a  tumour  evolves,  mutations  in  multiple  genes 
contribute  to  the  malignant  phenotype.  OVs 
specifically target cancer cells, because they are 
able to exploit the very same cellular defects that 
promote tumour growth.

Although  the  number  of  different  types  of  OVs 
that have been tested in preclinical trials is grow-
ing, only a few have made the transition into the 
clinic,  limiting  the  available  clinical  experience. 
These  new  therapeutics  are  therefore  still  the 
subject  of  research  as  the  scientific  community 
develops means to optimise their efficacy.

The THERADPOX project focuses on specific on-
colytic vectors such as pox virus and adenovirus-
based  vectors,  owing  to  their  inherent  strong 
oncolytic potencies and safety records. THERAD-
POX aims to improve the safety and therapeutic 
efficacy of OVs in vivo. 

Ultimately,  THERADPOX  targets  the  improve-
ment  of  cancer  treatment,  for  which  there  is  a 
high  unmet  medical  need  and  where  available 
effective  modalities  are  missing.  Owing  to  their 

inherent  strong  oncolytic  potencies  and  safety 
records, pox virus- and adenovirus-based vectors 
have been chosen as the vehicle for pursuing the 
goal of the project, which is to improve the safety 
and therapeutic efficacy of OVs in vivo. Novel and 
improved  OVs  will  be  engineered,  with  the  fol-
lowing traits in vivo: 

to  specifically  target  colorectal,  pan-

• 
creatic and ovarian cancer cells; 
• 
to replicate exclusively in cancer cells 
that  are  armed  with  therapeutic  genes 
rendering  only  tumour  cells  sensitive  to 
chemotherapy; 
• 
permit total tumour eradication. 

to widely spread within the tumour to 

Through  the  proposed  multidisciplinary  work 
plan  and  the  strong  and  complementary  ex-
pertise  of  the  10  European  partners  involved, 
THERADPOX will succeed in performing the fol-
lowing objectives: 

•  generate  advanced  knowledge  that 
could  be  translated  in  safer  cancer  treat-
ments  with  an  increased  therapeutic  in-
dex; 
• 
contribute to the improved quality of 
life  of  cancer  patients  by  offering  fewer 
treatments with no toxic side effects; 
•  propose new guidelines and standards 
for the development of OVs;
• 
rope in the war against cancer. 

strengthen the competitiveness of Eu-

NewTherapy_02.indd   107

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New Therapies – Gene Therapy

107

 
 
 
 
 
 
 
 
 
 
gENE THERAPY

THERAdPOX

Approach and methodology:

Common  strategies  and  methodologies 
were  defi ned  and  adapted  for  OVs  in  the 
THERADPOX  project.  The  establishment 
and  harmonisation  of  common  strategies 
related specifi cally to the following: vector 
engineering, production and purifi cation of 
oncolytic Pox and Ad; reporter genes; qual-
ity controls; standardisation of therapeutic 
potency  and  tumour  selectivity;  methods 
for tumour cell infection in vitro; common 
in  vitro  evaluation  of  the  functionality  of 
FCU1;  tumour  models;  and  standardised 
analysis of data (all partners).

Expected outcome:

It 
is  anticipated  that  THERADPOX  will 
achieve  the  development  of  oncolytic 
vectors  with  increased  tumour  selectivity 
of  infection,  replication  and  viral  spread 
through the tumour tissue that will subse-
quently enter clinical development.

Main fi ndings: 

Adenovirus and Pox viruses

Normal cells

Cancer cells

De-targeting

• Mutations viral surface proteins

Normal cells

Cancer cells

Targeting

• Surface expression of ligands which bind
to cancer cells receptors

Normal cells

Cancer cells

Replication

• Promoter mutations
• Promoter mutations
• Gene deletion

Normal cells

Cancer cells

Spreading

• Mutations
• Gene insertion
• Gene deletion

Arming

• Gene insertion: suicide FCU1 gene 

Tumour cell fusion

Normal 
cells

Cancer 
cells

Prodrug

Drug

Testing

The fi ndings of the project are set out be-
low:

Tumour cell lines
and biopsies in vitro

In vivo: Human tumours and
metastasis in mice

•  Enhanced tumour infectivity and 
tumour  selective  replication  con-
cerning  oncolytic  adenoviruses  through 
liver de-targeting and tumour targeting.
•  Engineering  of  various  optimised  on-
colytic adenoviruses. These chimeric viral 
capsids  were  constructed  in  such  a  way 
as  to  target  transcriptional  deregulated 
pathways in tumours; specifi cally, the E2F 
pathway  was  targeted  in  most  tumour 
types and the Wnt pathway in colon can-
cer  cells. These  engineered  adenoviruses 
also  exhibit  increased  replication  in  tu-
mour  cells  as  well  as  a  signifi cantly  im-
proved tumour-to-liver ratio in vitro and in 
vivo.

•  Confi rmed selective replication of the 
myxoma  virus  in  tumour  cells  and  their 
lack of replication in normal cells concern-
ing oncolytic Pox viruses. This set the basis 
for further molecular engineering of these 
viruses, so as to enhance their safety and 
selectivity  for  selective  tumour  cell  kill-
ing. Oncolytic vaccinia viruses were engi-
neered,  harbouring  gene  deletions,  with 
increased selectivity for tumour cells.
• 
Increased  spreading  of  an  oncolytic 
adenovirus  carrying  a  fusogenic  protein 
through  tumour  cells  in  culture,  aiming 
for  increased  spreading  of  OVs  through 
the tumour tissue. Oncolytic adenoviruses 

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New Therapies – Gene Therapy

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targeting  stromal  barriers  in  tumour  tis-
sue have also been engineered.
• 
“Armed” oncolytic adenoviruses with a 
gene coding for a non-toxic prodrug con-
verting enzyme, FCUI, targeting improved 
therapeutic efficacy of OVs.
•  Testing  of  various  formulations  to  al-
low  for  controlled  release  of  OVs  for  en-
hanced safety of OVs.
•  Selection of various tumour models for 
the in vivo testing of selected candidates.
•  Development  of  a  software  applica-
tion, allowing for a fast, sharp and stand-
ardised analysis of data. The website www.
theradpox.org will enable the dissemina-
tion of results to the scientific, clinical and 
citizen communities.

Conclusion:

Various  model  oncolytic  Pox  and  adenoviruses 
have  been  generated  as  proof  of  concept  to-
wards the development of oncolytic vectors with 
increased tumour selectivity of infection, replica-
tion and viral spread through the tumour tissue. 
The results obtained during the first year of the 
project  have  set  the  groundwork  for  the  devel-
opment of viral vectors with improved therapeu-
tic efficacy. Therefore, selected oncolytic pox and 
adenoviruses will be used to screen their tumour 
selectivity  in  a  panel  of  colorectal,  ovarian  and 
pancreatic tumour models during the period fol-
lowing the THERADPOX project.

gENE THERAPY

Coordinator

MonikaLusky
TRANSGENE SA
11 rue de Molsheim
67082 Strasbourg Cedex, France
E-mail: lusky@transgene.fr

Partners 

RamonAlemany
Institut Català d’Oncologia
Barcelona, Spain

StéphaneBertagnoli
Institut National de la Recherche Agronomique
Toulouse, France

RichardIggo
University of St Andrews
Bute Medical School
St Andrews, UK

HarryJalonen
DelSiTech Ltd 
Turku, Finland

AkseliHemminki
University of Helsinki
Helsinki, Finland

MohammedBenbouchaib
NewLab
Villers Les-Nancy, France

GerdSutter
Paul Ehrlich Institut
Langen, Germany

VictoriaSmith
Oncotest GmbH
Freiburg, Germany

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109

 
 
 
 
RIgHT

RNAinterferencetechnologyashumantherapeutictool

gENE THERAPY






ContractNo 
Projecttype

LSHB-CT-2004-00276
IntegratedProject

ECcontribution  e 11202230

Startingdate 1January200
Duration 48months

Website  www.ip-right.org/

Background and objectives:

The  research  initiative  RIGHT  aims  at  exploiting 
and further developing the vast potential of RNA 
interference  (RNAi)  to  provide  effective  thera-
peutic tools for the treatment of severe diseases, 
based  on  an  advanced  understanding  of  their 
underlying  mechanisms.RNA  interference  is  a 
naturally  occurring  mechanism  that  regulates 
gene expression and is naturally employed by the 
organism  as  defence  against  viruses.  Short  (21-
23  bp)  double-stranded  RNA  is  used  to  induce 
the sequence-specific degradation of mRNA and 
thereby block the synthesis of the corresponding 
protein. 

There  are  two  main  possibilities  to  apply  RNAi: 
small  interfering  RNAs  (siRNAs)  that  are  deliv-
ered  directly  into  cells  as  naked  siRNAs  or  with 
the help of a transfection reagent are used, while 
the other possibility is to use small hairpinRNAs 
(shRNA)  that  are  encoded  by  a  vector  and  de-
livered  into  the  cell,  and  are  thereby  expressed 
using  the  machinery  of  the  cell  itself.  As  a  new 
technology,  RNAi  has  revolutionised  basic  re-
search  by  enabling  gene  function  analysis  and 
by  providing  potential  new  therapeutic  strate-
gies.  Only  recently,  the  Nobel  Prize  in  Medicine 
was awarded to Craig C. Mello and Andrew Z. Fire 
for ‘RNA interference – gene silencing by double-
stranded RNA’.

Approach and methodology:

As a technique to control gene expression, RNAi 
has the potential to specifically regulate the pro-
duction of disease-associated genes. For applica-
tion  as  a  therapeutic  tool  in vivo,  it  is  necessary 
to overcome technical challenges, such as insuf-
ficient  uptake  or  low  stability  of  the  inhibitors, 
undesired  interferon  response  or  non-specific 
silencing  of  other  genes.  To  address  the  multi-
ple  facets  of  therapy  development,  the  RIGHT 
project  is  divided  into  5  competence  domains. 
Experts from various scientific fields are working 
together to exploit the vast potential of this in-
teresting  new  technology  and  to  make  the  ap-
plication  of  RNAi  as  a  therapeutic  tool  possible, 
in a multidisciplinary way.

110

New Therapies – Gene Therapy

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1)MolecularMechanismsandTechnologies:
The  understanding  of  the  molecular  processes 
associated with RNAi and the naturally occurring 
counterpart  microRNA  is  improved. This  knowl-
edge  serves  as  a  basis  for  the  development  of 
novel molecular strategies enabling the success-
ful  application  of  RNAi  technology  for  human 
therapy. Use is made of large-scale RNAi libraries 
and  high-throughput  screening  technologies 
to identify new target genes for therapeutic ap-
proaches and to analyse identified inhibitors.

2)ChemicalTools:
New chemically modified siRNAs are synthesised 
and  extensively  tested  in  cell  culture  and  living 
organisms in order to increase sensitivity, specifi-
city, delivery, stability and cost-effectiveness, and 
to reduce side effects.

3)GeneticTools:
Potent  viral  or  non-viral  RNAi  delivery  vectors 
are  generated  and  their  features  evaluated  in 
relation  to  their  chemical  counterparts.  Special 
emphasis is given to the development of tissue-
specific and inducible systems.

4)Pharmacokinetics:
For the development of a drug, synthetic or ge-
netic  RNAi,  reagents  are  tested  in  cultured  cells 

gENE THERAPY

Overview of the five competence domains in RIGHT

with  pharmacokinetic  methods.  Successful  can-
didates are then assessed in animal models, and 
extensive phenotyping of treated animals will be 
performed.

)CellBiologyandDiseaseModels:
Selected  disease  models  are  used  for  the  para-
digmatic  assessment  of  RNAi  as  a  therapeutic 
tool. This generates RNAi leads for clinical tests. In 
particular,  RIGHT  focuses  on  infectious  diseases 
and  genetic  defects  causing  degenerative  dis-
eases and cancer.

Expected outcome:

Within 4 years, the potential of RNAi to diagnose 
and  successfully  treat  diseases  will  be  demon-
strated  and  proof  of  principle  will  be  provided 
for the value of RNAi as a therapeutic tool in liv-
ing organisms.

Main findings:

In the RIGHT project, new tools for the application 
and evaluation of RNAi, such as random libraries 
or high throughput techniques have been devel-
oped.  New  microRNAs  could  be  identified  and 
their  function  analysed  in  more  detail.  It  could 
be  shown  that  miR-181  participates  in  muscle 

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New Therapies – Gene Therapy

111

gENE THERAPY

RIgHT

models, like influenza and HBV. For these disease 
models, specific inhibitors were identified in vitro. 
These inhibitors will now be optimised for appli-
cation  in vivo  and  their  effects  tested  in  animal 
models.

Major publications

Piva, R., Pellegrino, E., Mattioli, M., Agnelli, L., Lom-
bardi,  L.,  Boccalatte,  F.,  Costa,  G.,  Ruggeri,  B.A., 
Cheng,  M.,  Chiarle,  R.,  Palestro,  G.,  Neri,  A.,  Ing-
hirami, G., ‘Functional validation of the anaplastic 
lymphoma kinase signature identifies CEBPB and 
BCL2A1 as critical target genes’, J Clin Invest, 2006, 
116(12):3171-82.

Pal, A., Severin, F., Lommer, B., Shevchenko, A., Ze-
rial,  M.,  ‘Huntingtin-HAP40  complex  is  a  novel 
Rab5 effector that regulates early endosome mo-
tility and is up-regulated in Huntington’s disease’, 
J. Cell Biol, 2006, 13;172(4):605-18.

Brown,  B.D., Venneri,  M.A.,  Zingale,  A.,  Sergi,  L.S., 
Naldini,  L.,  ‘Endogenous  microRNA  regulation 
suppresses transgene expression in haematopoi-
etic  lineages  and  enables  stable  gene  transfer’, 
Nat Med, 2006, 12(5):585-91.

Naguibneva, I., Ameyar-Zazoua, M., Polesskaya, A., 
Ait-Si-Ali, S., Groisman, R., Souidi, M., Cuvellier, S., 
Harel-Bellan,  A., ‘The  microRNA  miR-181  targets 
the homeobox protein Hox-A11 during mamma-
lian  myoblast  differentiation’,  Nat  Cell  Biol,  2006, 
8(3):278-84.

Varghese,  O.P.,  Barman,  J.,  Pathmasiri,  W.,  Plas-
hkevych,  O.,  Honcharenko,  D.,  Chattopadhyaya, 
J.,  ‘Conformationally  constrained  2’-N,4’-C-eth-
ylene-bridged  thymidine  (aza-ENA-T):  synthe-
sis,  structure,  physical,  and  biochemical  studies 
of  aza-ENA-T-modified  oligonucleotides’,  J  Am 
Chem Soc, 2006, 128(47):15173-87.

Casares, N., Pequignot, M.O., Tesniere, A., Ghiring-
helli, F., Roux, S., Chaput, N., Schmitt, E., Hamai, A., 

Press conference at Max Planck Institute in Berlin, from left 
to right: Prof. Dr. Joachim W. Engels, Prof. Dr. Thomas F. Meyer, 
Prof. Dr. Arndt Borkhardt

cell differentiation (Naguibneva et al. 2005) and 
that  miR-223  is  involved  in  human  granulocyte 
differentiation (Fazi et al., 2005). 

In  the  field  of  chemically-modified  siRNAs,  the 
invention  of  a  new  design  for  these  inhibitors 
shows  promising  results. This  new  design  leads 
to higher stability of the inhibitors and allows ad-
ditional modifications that are under evaluation 
in  the  consortium.  These  modifications  could 
help to increase targeting and reduce undesired 
side effects of the compounds. This new design 
has already been submitted for patenting.

Besides the construction of synthetic inhibitors, 
efforts  are  undertaken  to  develop  new  vector 
systems for the expression of shRNAs. Here, some 
first  promising  results  show  the  possibility  to 
use lentiviral vectors that include miRNA target 
sequences to regulate gene expression in differ-
ent  organs  (Brown  et  al.,  2006). With  these  vec-
tors,  the  delivery  and  segregated  expression  of 
shRNAs  among  different  tissues  is  possible  and 
should provide a new tool for  more efficient and 
safe expression of shRNAs.

In the first two years of the project several disease 
models were established to assess the potential 
of RNAi approaches in vivo. These models include 
different  cancer  models  and  infectious  disease 

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gENE THERAPY

Coordinator 

ThomasF.Meyer
Max-Planck-Institute for Infection Biology
Charitéplatz 1
D 10117 Berlin, Germany
E-mail: right@mpiib-berlin.mpg.de

Partners

ThomasRudel
Max Planck Institute for Infection Biology
Berlin, Germany

JørgenKjems
University of Aarhus
Aarhus, Denmark

OlliKallioniemi
VTT Technical Research Centre of Finland
Turku, Finland

JesperWengel
University of Southern Denmark
Odense, Denmark

JyotiChattopadhyaya
University of Uppsala
Uppsala, Sweden

AnnickHarel-Bellan
Institut André Lwoff, CNRS
Villejuif, France

CarolaPonzettoandGiorgioInghirami
University of Torino
Turin, Italy

MarinoZerial
Max Planck Institute of Molecular Cell Biology and 
Genetics
Dresden, Germany

Poster from a public symposium organised by RIGHT in 
Paris, Institut Pasteur in October 2006

Hervas-Stubbs, S., Obeid, M., Coutant, F., Métivier, 
D., Pichard, E., Aucouturier, P., Pierron, G., Garrido, 
C.,  Zitvogel,  L.,  Kroemer,  G., ‘Caspase-dependent 
immunogenicity  of  doxorubicin-induced  tumor 
cell death’, J Exp Med, 2005, 19;202(12):1691-701.

Fazi, F., Rosa, A., Fatica, A., Gelmetti, V., De Marchis, 
M.L.,  Nervi,  C.,  Bozzoni,  I.,  ‘A  minicircuitry  com-
prised of microRNA-223 and transcription factors 
NFI-A and C/EBPalpha regulates human granulo-
poiesis’, Cell, 2005, 123(5):819-31.

Patents

One patent application was submitted within the 
RIGHT project by Jesper Wengel (Southern Den-
mark  University)  and  Jørgen  Kjems  (University 
of Aarhus) on a new design for small interfering 
RNAs (Danish Patent Application 2006 00433).

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113

gENE THERAPY

RIgHT

MichaelRoberts
Regulon
Athens, Greece

JoachimEngels
Johann Wolfgang Goethe-Universität
Frankfurt, Germany

PatrickBrianArbuthnot
University of the Witwatersrand
Johannesburg, South Africa

GuidoKroemer
INSERM
Villejuif, France

PietHerdewijn
Katholieke Universiteit Leuven
Leuven, Belgium

ArndtBorkhardt
Heinrich-Heine- Universität Düsseldorf - Universitätsk-
linikum
Düsseldorf, Germany

LuigiNaldini
Fondazione Centro San Raffaele del Monte Tabor
Milano, Italy

WlodzimierzKrzyzosiak
Polish Academy of Sciences
Poznan, Poland

JörnGlökler
RiNA GmbH
Berlin, Germany

IreneBozzoni
University of Rome “La Sapienza”
Rome Italy

JohanAuwerx
Insitut Clinique de la Souris (ICS)
Illkirch, France

GeorgeMosialosandGeorgeKollias
Biomedical Sciences Research Centre “Al. Fleming”
Vari (Attica), Greece

PatrickErbacher
PolyPlus-transfection SA
Illkirch, France

SaraSkogsäter
ARTTIC
Paris, France

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ZNIP

Therapeuticin vivoDNArepairbysite-specificdouble-strandbreaks

ContractNo 
Projecttype

LSHB-CT-2006-037783
SME-SpecificTargetedResearchProject

ECcontribution  e 2349996

Startingdate 1January2007
Duration 36months






Background and objectives:

To date, more that 6 000 human single gene dis-
orders have been identified, affecting about 2% 
of  the  population. To  cure  or  prevent  disorders 
caused  by  single  gene  defects,  direct  in  situ  re-
pair would be an attractive strategy, particularly 
for  diseases  where  the  therapeutic  approach 
could be based on short sequence substitutions, 
deletions or insertions. 

Currently,  most  gene  therapy  approaches  are 
based  on  the  delivery  of  a  therapeutic  gene 
coupled  to  promoter  sequences.  Some  of  the 
therapeutic sequences may be replacements for 
a mutated gene, while other approaches aim to 
kill  or  protect  particular  cells.  However,  the  de-
livery of genes that are taken from their natural 
context poses a number of problems and limita-
tions.  Thus,  a  strategy  that  corrects,  or  disrupts, 
a  gene  in  situ  is  a  central  biomedical  challenge 
that  unlocks  entirely  new  strategies  for  func-
tional genomics, and ultimately, for gene therapy. 
Direct and site-specific modification of a gene at 
its natural locus, without the introduction of ad-
ditional sequences like selection cassettes, offers 
an appealing novel strategy in gene therapy. 

Such gene targeting is based upon the cells’ ca-
pacity  to  carry  out  homologous  recombination 
(HR), a process which results in an accurate and 
precise  exchange  of  genetic  material  between 
the introduced donor DNA and the homologous 
genomic  target  DNA.  The  efficiency  of  conven-

tional  gene  targeting  by  HR  increases  with  the 
length  of  homology  between  the  donor  DNA 
and the target sequence. Although conventional 
gene targeting is an invaluable tool for creating 
transgenic  laboratory  animals,  the  technique  is 
hampered  by  major  limitations,  including  low 
targeting  frequencies,  generally  with  only  one 
targeted cell in every 105-107 treated cells. 

Moreover, in most mammalian cells, the ratio be-
tween  site-specific  homologous  recombination 
and non-homologous end joining (NHEJ) is un-
favourable. Consequently, until now, gene target-
ing has depended on sensitive selection systems 
in order to identify the few cells where gene tar-
geting  has  occurred.  Such  selection  strategies 
normally require the donor DNA to contain extra 
non-homologous  sequences  coding  for  selec-
tion markers (often derived from bacteria or vi-
ruses). These can result in undesired side effects 
on the cells, as exemplified by down regulation 
of expression due to de novo methylation.

Designed endonucleases have recently provided 
a  revolutionary  tool  for  achieving  high  rates  of 
targeted  genome  alteration.  By  stimulating  ho-
mologous  recombination  through  the  creation 
of site-specific targeted double strand breaks, in 
vivo sequence alteration rates can be obtained. 

In ZNIP, a major effort is in progress to generate 
tools, both improved zinc finger endonucleases 
and  second  generation  TFOs  (Triplex-forming 
oligonucleotides)  for  targeted  sequence  altera-

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New Therapies – Gene Therapy

11

 
 
gENE THERAPY

ZNIP

encounter  DNA  damage  that  challenges  their 
genomic  integrity,  they  generally  activate  DNA 
repair pathways and cell cycle checkpoint signal-
ling in order to counteract the damage. Moreo-
ver, if the DNA damage can not be repaired, cells 
can activate apoptotic signalling pathways. 

Finally, the technology will be tested in primary 
stem cells and transgenic reporter mice as an im-
portant step towards clinical trials. 

Approach and methodology:

ZNIP will proceed by the following steps:

computer-assisted  design  of  new  en-
• 
donuclease  specificities  and  in  vitro  vali-
dation; 
•  development  of  new  bioassays  for  in 
vivo  optimisation  of  zinc  finger  endonu-
cleases;
•  analysis  of  pathways  involved  in  tar-
geted  sequence  correction  with  DNA 
double strand breaks; 
•  application  of  these  pathways  for 
improvement  and  increase  of  targeting 
rates;
•  analysis  of  the  cellular  responses  to 
targeted sequence alteration: aberrant in-
tegration and toxicity;
•  analysis  of  targeted  sequence  correc-
tion  in  animal  models  and  natural  stem 
cell populations; 
•  analysis and improvement of delivery 
and dose control.

Expected outcome:

ZNIP anticipates further development of a novel 
technique for targeted sequence alteration in liv-
ing cells as a tool for gene therapy. Designed en-
donucleases are a revolutionary new tool for in-
ducing site-specific genome alterations. They will 
have a significant impact on future gene therapy 
protocols, as well as on a broad spectrum of bio-
technological applications.

tion in living cells. The goal of the ZNIP consor-
tium  is  to  take  gene  repair  protocols  to  a  level 
where high rates can be obtained with consist-
ency, high efficacy and specificity, as well as with 
strongly reduced levels of side effects. To achieve 
this goal, the consortium will proceed as follows: 
improve  and  test  sequence-specific 

• 
DNA zinc finger endonucleases (ZFNs); 
•  develop  triple  helix-forming  oligonu-
cleotides (TFOs) that induce high rates; 
•  elucidate  and  constructively  improve 
the  cellular  mechanisms  involved  in  the 
process; 
• 
test  the  targeted  sequence  alteration 
in primary cells (including stem cells) and 
in living test animals.

Only  limited  knowledge  is  available  on  the  cel-
lular pathways and proteins that are involved in 
and/or  regulate  targeted  gene  correction  after 
site-specific  DNA  double  strand  breaks.  A  bal-
ance between HR and NHEJ may determine the 
efficiency  of  gene  correction.  However,  other 
pathways may also be involved. To improve tar-
geting  frequencies  and  targeting  safety  (i.e.  to 
reduce  genotoxic  side-effects  by  non-homolo-
gous end-joining, for instance), it is of paramount 
importance to understand the role of cellular fac-
tors  in  the  correcting  pathway(s);  this  work  will 
be carried out extensively by the consortium.

Furthermore, it is important to establish whether 
cellular pathways that could impair the viability 
of  the  targeted  cells  are  activated.  When  cells 

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gENE THERAPY

RalfWagner
GENEART GmbH
D-93053 Regensburg, Germany 

Coordinator

StefanKrauss
Rikshospitalet
Section for cellular and genetic therapy
Forskningparken
Gaustadalleen 21
0349 Oslo, Norway
E-mail: Stefan.krauss@medisin.uio.no

Partners

ToniCathomen
Institute of Virology
Charité Universitätsmedizin Berlin
Campus Benjamin Franklin
Berlin, Germany 

KlausSchwarz
University Hospital Ulm
Transfusion Medicine
Ulm, Germany 

LuisSerrano
Centre de Regulació Genomica
Barcelona, Spain

TomBrown
University of Southampton
School of Chemistry
Highfield, Southampton, UK 

RolandKanaar
Erasmus Medical Centre
Department of Cell Biology and Genetics
Rotterdam, Netherlands

BengtNordén
Chalmers University of Technology
Physical Chemistry
Gothenburg, Sweden 

ATDBioLtd
Highfield, Southampton, UK 

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117

SNIPER

Sequencespecificoligomersforin vivoDNArepair

gENE THERAPY

ContractNo 
Projecttype

LSHB-CT-2004-00204
SpecificTargetedResearchProject

ECcontribution  e 203000

Startingdate 1January200
Duration 36months






Background and objectives:

Gene targeting can be defined as a method for 
introducing site-specific sequence alterations in 
the genomes of living cells. Successful gene tar-
geting would have far reaching implications for 
the construction of mutant cell lines and animals, 
both  for  studies  of  gene  function  as  well  as  for 
the  development  of  disease  models.  Further-
more, direct modification of a target gene at its 
genomic  location,  without  the  introduction  of 
additional redundant sequences, would offer an 
appealing strategy for gene therapy. 

Use  of  gene  targeting  in  order  to  create  trans-
genic cell lines does not necessarily require high 
targeting rates as long as powerful selection bio-
essays can be applied to facilitate isolation of the 
targeted cells. However, for a clinical application 
in  gene  therapy  protocols,  the  introduction  of 
selective markers in the target genome is not de-
sirable and, consequently, such strategies require 
higher  targeting  rates.  For  example,  it  has  been 
estimated that in order to observe a beneficial ef-
fect from the correction of the mutation under-
lying  the  monogenetic  disease  haemophilia,  at 
least 1%-5% of the treated cells need to express 
the corrected protein. 

Since targeted sequence alteration in living cells 
is increasingly turning into a promising tool for 
gene therapy applications, it is important to ana-
lyse  the  various  strategies  needed  for  site  spe-
cific  DNA  alterations.  The  SNIPER  consortium  is 

focusing on strategies based on singles stranded 
oligonucleotides  (ssODN)  and  triple  helix  form-
ing oligonucleotides (TFO).

TFOs have demonstrated genome altering activ-
ity  in  vitro  and  in  vivo.  Improving  selective  site-
specific  DNA  recognition  by  TFOs  is  a  central 
requirement, because the lack of a general recog-
nition code restricts TFO applicability to a limited 
number  of  loci  in  the  genome. The  consortium 
is working towards modified bases in the triple-
helix-forming  oligo-  nucleotides  that  tolerate 
pyrimidine  interruptions  and  that  contribute  to 
increased triplex stability and binding kinetics. 

A  second  class  of  molecules  capable  of  se-
quence-specific DNA recognition is the peptide 
nucleic acids (PNAs). PNAs can recognise DNA by 
three modes: triplex invasion; duplex invasion; or 
double duplex invasion. Of these, the triplex and 
double  duplex  invasion  approaches  are  of  par-
ticular interest for targeted gene repair and are 
being  analysed  by  the  consortium.  Using  vari-
ous chemical modifications, the project partners 
have improved DNA binding properties of TFOs 
and PNAs. The consortium has also strongly im-
proved the properties of the sequence template 
for the targeted repair reaction by a combination 
of structural improvements and a novel strategy 
based on an active group transfer.

By using different mechanisms, ssODN have the 
potential  to  achieve  site-specific  alterations  in 
the sequence of a genomic DNA template in liv-

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gENE THERAPY

improved  cellular  delivery 

in the observed targeted repair;
•  develop 
systems; 
•  provide a thorough analysis of poten-
tial side effects.

Expected outcome:

The  project  will  further  develop  strategies  for 
targeted  sequence  alteration  in  living  cells  as  a 
tool for biotechnology and gene therapy.

Main findings:

High rate site-specific targeted sequence altera-
tion is likely to prove a viable strategy. Strategies 
based on a combination of site specific endonu-
cleases and selected DNA templates are offering 
substantial  promise  for  future  biotechnological 
and gene therapy applications. 

Major publications

Olsen, P.A., Randol, M., Luna, L., Brown, T., Krauss, S., 
‘Genomic sequence correction by single-strand-
ed DNA oligonucleotides: role of DNA synthesis 
and  chemical  modifications  of  the  oligonucle-
otide ends’, J Gene Med, 2005, Dec;7 (12):1534-44.

Olsen, P.A., Randol, M., Krauss, S., ‘Implications of 
cell  cycle  progression  on  functional  sequence 
correction  by  short  single-stranded  DNA  oligo-
nucleotides’, Gene Ther, 2005, 12:546-51.

Fox, K.R., Brown, T., ‘An extra dimension in nucleic 
acid  sequence  recognition’,  Q Rev Biophys,  2005, 
Nov;38 (4):311-20. 

Alzeer,  J.,  Scharer,  O.D., ‘A  modified  thymine  for 
the  synthesis  of  site-specific  thymine-guanine 
DNA  interstrand  crosslinks’,  Nucleic  Acids  Res, 
2006, 34(16):4458-66. 

Frykholm, K., Morimatsu, K., Norden, B., ‘Conserved 
conformation of RecA protein after executing the 

ing cells. The consortium has shown that ssODN 
induce significant rates of in vivo sequence cor-
rection rates.

A critical aspect for the further development of 
gene repair approaches is the elucidation of the 
cellular  responses  needed  to  achieve  targeted 
sequence  alteration.  For  this  purpose,  siRNA-
based screens are carried out in order to identify 
factors  involved  in  gene  repair. The  consortium 
also seeks to gain an undestanding of the cellu-
lar components of the process by studying those 
components that make up the cellular mismatch 
repair machinery, as well as those  of the replica-
tion machinery and  of the cell cycle checkpoints. 
The  consortium  successfully  demonstrated  the 
role of the cell cycle on in vivo targeted sequence 
alteration. 

Despite  the  substantial  challenges  that  remain, 
targeted  sequence  alteration  based  on  ssODN 
promises to become a part of the standard toolkit 
for functional gene alterations.

Approach and methodology:

SNIPER aims to:

improve  platform  technology  for  tar-
• 
geted sequence alteration in living viable 
cells using single strand oligonucleotides;
• 
improve  TFO  modules  by  chemical 
modifications; 
•  design  and  implement  new  reactions 
to induce single base pair alterations; 
•  define  the  cellular  pathways  involved 

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New Therapies – Gene Therapy

119

 
 
 
 
 
 
gENE THERAPY

SNIPER

DNA  strand-exchange  reaction.  A  site-specific 
linear dichroism structure study’,
Biochemistry, 2006, Sep 19;45(37):11172-8.

Hanada, K., Budzowska, M., Modesti, M., Maas, A., 
Wyman,  C.,  Essers,  J.,  Kanaar,  R.,  ‘The  structure-
specific  endonuclease  Mus81-Eme1  promotes 
conversion  of  interstrand  DNA  crosslinks  into 
double-strands breaks’, EMBO J, 2006, 25:4921-32. 

Li, H., Broughton-Head, V.J., Peng, G., Powers, V.E., 
Ovens, M.J., Fox, K.R., Brown, T.,
‘Triplex staples: DNA double-strand cross-linking 
at internal and terminal sites using psoralen-con-
taining triplex-forming oligonucleotides’, Biocon-
jug Chem, 2006, Nov-Dec;17 (6):1561-7.

Wyman, C., Kanaar, R., ‘DNA double-strand break 
repair:  all’s  well  that  ends  well’,  Annu  Rev  Genet, 
2006; 40:363-83.

Kim, K.H., Fan, X.J., Nielsen, P.E., ‘Efficient Sequence-
Directed  Psoralen  Targeting  Using  Pseudocom-
plementary  Peptide  Nucleic  Acids’,  Bioconjug 
Chem, 2007, Jan 26.

Coordinator

StefanKrauss
Rikshospitalet
Section for cellular and genetic therapy
Forskningparken
Gaustadalleen 21
0349 Oslo, Norway
E-mail: Stefan.krauss@medisin.uio.no

Partners

UlfEllervik
Lund Institute of Technology
Lund University
Lund, Sweden



Tom Brown
University of Southampton
Highfield, Southampton, UK 

KeithR.Fox
School of Biological Sciences
University of Southampton
Southampton, UK

RolandKanaar
Erasmus Medical Centre
Rotterdam, Netherlands 

BengtNordén
Chalmers University of Technology
Gothenburg, Sweden 

PeterE.Nielsen
University of Copenhagen
Copenhagen, Denmark

OrlandoD.Schärer
Institute of Molecular Cancer Research
University of Zurich
Zurich, Switzerland

DorcasBrown
ATDBio Ltd
Highfield, Southampton, UK. 

RalfWagner
GENEART GmbH
Regensburg, Germany

120

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gENE THERAPY

IMPROvEd PRECISION

Improvedprecisionofnucleicacidbasedtherapyofcysticfibrosis






ContractNo 
Projecttype

LHSB-CT-2004-00213
SpecificTargetedResearchProject

ECcontribution  e 304000

Startingdate 1December2004

Duration 26months

Website  www.improvedprecision.com

Background and objectives:

Cystic  fibrosis  (CF)  is  a  recessive  congenital  dis-
ease with a high incidence in Caucasian popula-
tions (1 in 3 000 newborns is affected). It is caused 
by mutations in the gene coding for CFTR (cystic 
fibrosis transmembrane conductance regulator), 
a  chloride  channel,  which  results  in  decreased 
chloride  secretion  and  hyper  absorption  of  so-
dium across epithelia. These disturbances in ion 
fluxes result in water hyper-absorption from the 
airway  surface  liquid  into  the  cells,  which  pro-
duce  a  highly  viscous  and  elastic  mucus. These 
changes  result  in  chronic  bacterial  infection  of 
the airways, leading to chronic lung disease.  

Despite recent developments in the treatment of 
CF there is no definite cure for this disease, and 
life expectancy is around 30 years. Several obser-
vations  suggest  that  a  downregulation  of  ENaC 
restores the perciliary liquid layer, thereby rehy-
drating  the  mucus  and  improving  ciliary  clear-
ance in the lung. Therefore, the members of the 
consortium  propose  to  specifically  downregu-
late ENaC expression by RNA interference.

The  intention  is  to  develop  compounds  able  to 
modulate ENaC expression, either transiently by 
synthetic siRNA constructs or plasmids express-
ing  siRNA  constructs,  or  long-term  by  lentiviral 
gene  transfer  for  stable  chromosomal  integra-
tion,  or  minichromosomes  expressing  siRNA 
constructs.  After  testing  these  compounds  in 
murine  cell  lines,  the  consortium  members  will 

use optimised compounds in established foetal 
and adult ΔF 508del mice and in a new (β-ENaC 
overexpressing transgenic mouse) animal model 
of cystic fibrosis.

Approach and methodology:

This comprehensive approach of interfering with 
ENaC expression by delivering siRNA to the air-
ways, includes the following objectives:
1.  Develop  compounds  able  to  modulate  ENaC 
expression by RNA interference:

•  novel formulations of synthetic siRNA 
and plasmid expression systems,
•  minichromosomes for expressing siRNA,
lentiviral vectors for expressing siRNA;
• 
2.  Establish  aerosolisation  of  such  compounds 
without damaging the compounds.
3.  Formulate  such  compounds  as  magnetic 
vectors, in order to make them susceptible to  
magnetic field guidance (“Magnetofection” tech-
nology)  and  combine  the  magnetic  approach 
with aerosolisation.
4. Develop instrumentation to generate magnet-
ic gradient fields for magnetic lung targeting.
.  Evaluate  and  validate  the  novel  formulations 
and  delivery  technologies  using  meaningful 
models in vitro and in vivo.

Expected outcome:

The expected outcome of this project is to dem-
onstrate  that  the  downregulation  of  ENaC  sub-
units  by  RNA  interference  in  airway  epithelial 

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121

 
 
 
 
 
 
gENE THERAPY

IMPROvEd PRECISION

Xenariou,  S.,  Griesenbach,  U.,  Ferrari,  S.,  Dean,  P., 
Scheule, R.K., Cheng, S.H., Geddes, D.M., Plank, C., 
Alton,  E.W.,  ‘Using  magnetic  forces  to  enhance 
non-viral  gene  transfer  to  airway  epithelium  in 
vivo’, Gene Ther, 2006.

Babincova,  M.,  Babinec,  P.,  ‘Aerosolized  VEGF  in 
combination with intravenous magnetically tar-
geted  delivery  of  DNA-nanoparticle  complex 
may  increase  efficiency  of  cystic  fibrosis  gene 
therapy’ Med Hypotheses, 2006, 67(4):1002. 

Schillinger, U., Brill, T., Rudolph, C., Huth, S., Gerst-
ing,  S.,  Krotz,  F.,  Hirschberger,  J.,  Bergemann,  C., 
Plank, C., ‘Advances in magnetofection - magneti-
cally guided nucleic acid delivery’, Journal of Mag-
netism and Magnetic Materials, 2005, 293(1):501-
508.

Rudolph,  C.,  Ortiz,  A.,  Schillinger,  U.,  Jauernig,  J., 
Plank, C., Rosenecker, J., ‘Methodological optimi-
zation of polyethylenimine (PEI)-based gene de-
livery to the lungs of mice via aerosol application’, 
Journal of Gene Medicine, 2005, 7(1):59-66.

Rudolph, C., Schillinger, U., Ortiz, A., Plank, C., Go-
las, M.M., Sander, B., Stark, H., Rosenecker, J., ‘Aero-
solized  nanogram  quantities  of  plasmid  DNA 
mediate highly efficient gene delivery to mouse 
airway  epithelium’,  Molecular  Therapy,  2005, 
12(3):493-501.

cells  in vitro  and  in vivo  is  feasible.  Evidence  for 
efficient  downregulation  should  be  shown  on 
the level of mRNA- and protein concentration of 
the  three  ENaC  subunits:  α,-β,-,  and  γ-.  In  addi-
tion, a correction of the pathologic sodium hyper 
absorption which characterises cystic fibrosis air-
way epithelia is expected by downregulation of 
ENaC-subunits.  For the in vivo experiments, a CF-
mouse model, as well as β -ENaC-overexpressing 
transgenic  mouse  model,  will  be  tested.  At  the 
end  of  the  project,  enough  evidence  should  be 
available to initiate a clinical trial on RNA interfer-
ence for cystic fibrosis lung disease by targeting 
ENaC.

Main findings:

Downregulation  of  ENaC  mRNA  and  sodium 
transport  function  in  cell  culture  systems  re-
sulting  in  significant  electrophysiologic  effects 
on  ENaC-mediated  sodium  uptake,  could  be 
achieved by RNA interference. Currently, experi-
ments in animal models are ongoing to confirm 
these data. 

OZ Biosciences, a SME partner in this project, as 
in this project, commercialised three novel mag-
netic  nanoparticle  formulations  based  on  Mag-
netofection  (SilenceMag,  ViroMag  and  ViroMag 
R/L), and one lipid-based formulation specific to 
siRNA  applications  (Lullaby®  siRNA  transfection 
reagent).  

Major publications 

Mykhaylyk, O., Vlaskou, D., Tresilwised, N., Pithay-
anukul, P., Moller, W., Plank, C., ‘Magnetic nanopar-
ticle  formulations  for  DNA  and  siRNA  delivery’,  
Journal  of  Magnetism  and  Magnetic  Materials, 
2007, in press.

Dames, P., Ortiz, A., Schillinger, U., Lesina, E., Plank, 
C., Rosenecker, J., Rudolph, C.,  ‘Aerosol gene de-
livery to the murine lung is mouse strain depend-
ent’, J Mol Med, 2006, Dec 8.

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gENE THERAPY

HolgerSchankinandHansSchreier
MCS Micro Carrier Systems GmbH
Neuss, Germany

OlgaZegarra-Moran
Laboratorio di Genetica Molecolare
Istituto Giannina Gaslini
Genoa, Italy

PeterBabinecandMelaniaBabincova
Comenius University
Department of Nuclear Physics and Biophysics 
Bratislava, Slovakia

DetlefEricHinzandLenaGrimm
Fraunhofer Patent Centre                           
Munich, Germany

Coordinator 

JosephRosenecker
Klinikum der Universität München 
Kinderklinik und Kinderpoliklinik im Dr. von Haunerschen 
Kinderspital
Lindwurmstraße 4  
D-80337 München, Germany
E-mail: Joseph.Rosenecker@med.uni-muenchen.de 

Partners

MassimoConese,ElenaCopreni
San Raffaele University
Institute for Experimental Treatment of Cystic Fibrosis 
- DIBIT
Milan, Italy

FiorentinaAscenzioni
University of Rome “La Sapienza”
Dipartimento di Biologia Cellulare e dello Sviluppo
Rome, Italy

OlivierZelphati
OZ Biosciences 
Parc Scientifique et Technologique de Marseille-Luminy 
Marseille, France 

CharlesCoutelleandSuzyBuckley
Imperial College London                                        
Faculty of Medicine                                     
Division of Biomedical Sciences                          
Gene Therapy Research Group                            
London, UK

ChristianPlankandOlgaMykhaylyk
Institut für Experimentelle Onkologie der TUM
Klinikum r. d. Isar
Munich, Germany

BobJ.Scholte,PatriciaSpijkers
Erasmus Medical Centre
Cell Biology Department
Rotterdam, Netherlands

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123

INTHER

Development and application of transposons and site-specific
technologiesasnon-viralgenedeliverymethodsforevvivogene-basedtherapies

integration

gENE THERAPY






ContractNo 
Projecttype

LSHB-CT-200-018961
SpecificTargetedResearchProject

ECcontribution  e 2800000

Startingdate 1November200

Duration 36months

Website  www.mdc-berlin.de/izsvak/eng

Background and objectives:

Considerable  effort  has  been  devoted  to  the 
development of gene delivery strategies for the 
treatment of inherited and acquired disorders in 
humans. Ex vivo gene therapies are based on re-
moving cells from a patient, introducing a thera-
peutic gene construct into the cells, and implant-
ing the engineered cells back into the patient. Ex 
vivo gene therapy is less cost-effective and more 
labour-intensive  than  in  vivo  gene  therapy,  but 
from  a  safety  perspective,  it  is  potentially  more 
attractive. 

Currently,  both  viral  and  non-viral  methods  are 
used  for  gene  delivery.  Adapting  viruses  for 
gene  transfer  is  a  popular  approach,  but  safety, 
immunogenicity and production issues hamper 
clinical progress. The establishment of non-viral, 
integrating  vectors  generated  considerable  in-
terest in developing efficient and safe vectors for 
human gene therapy. 

Transposable elements are recombinases that can 
be considered as natural, non-viral delivery vehi-
cles,  capable  of  efficient  genomic  insertion.  The 
use of transposable elements can address one of 
the main problems of non-viral technologies: sta-
ble  genomic  integration  provides  long-term  ex-
pression  of  therapeutic  genes.  Using  transposon 
vectors, integration from plasmid DNA into chro-
mosomes provides the basis for long-term expres-
sion of therapeutic genes in treated cells. 

This  project  will  evaluate  the  available  recombi-
nase systems in terms of their efficacy and safety 
features.  INTHER  proposes  to  establish  optimal 
delivery  protocols  for  the  therapeutic/recombi-
nase-systems  into  a  variety  of  cells.  It  will  deter-
mine what type of cells and diseases models are 
best  suited  to  the  recombinase  technology  that 
can offer an alternative to existing viral and non-
viral technologies.

Approach and methodology:

INTHER  aims  to  establish  and  apply  methods 
and  protocols  for  efficient  nucleic  acid/protein 
delivery into therapeutically relevant target cells, 
in the context of recombinase technologies. The 
overall objective of INTHER is to develop non-vi-
ral,  recombinase-based  technologies  for  ex  vivo 
gene  therapeutic  applications  as  an  alternative 
to current viral/non-viral gene delivery technolo-
gies,  with  the  aim  of  circumventing  the  toxicity 
and  immunogenicity  problems  raised  by  viral 
delivery systems. 

Novel  gene  transfer  technologies  will  be  estab-
lished  by  developing  transposon  vectors  that 
mediate  efficient  and  targeted  integration  of 
therapeutic genes into the genome. INTHER aims 
at  developing  recombinases  tailored  for  gene 
therapeutic  purposes,  by  using  three  different 
recombinase  systems:  SB,  FP  and  PhiC31.  The 
project will evaluate which recombinase system 
holds the greatest promise for further develop-
ment, by performing head-to-head comparisons 
among the three recombinase systems.

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gENE THERAPY

Figure 1. Experimental strategies for targeting Sleeping 
Beauty transposition. The common components of the tar-
geting systems include a transposable element that contains 
the IRs (arrowheads) and a gene of interest equipped with 
a suitable promoter. The transposase (purple circle) binds to 
the IRs and catalyzes transposition. A DNA-binding protein 
domain (red oval) recognizes a specific sequence (turquoise 
box) in the target DNA (parallel lines). (a) Targeting with 
transposase fusion proteins. Targeting is achieved by fusing 
a specific DNA-binding protein domain to the transposase. 
(b) Targeting with fusion proteins that bind the transposon 
DNA. Targeting is achieved by fusing a specific DNA-binding 
protein domain to another protein (white oval) that binds to 
a specific DNA sequence within the transposable element 
(yellow box). In this strategy, the transposase is not modified. 
(c) Targeting with fusion proteins that interact with the 
transposase. Targeting is achieved by fusing a specific DNA-
binding protein domain to another protein (light green oval) 
that interacts with the transposase. In this strategy, neither 
the transposase nor the transposon is modified.

Figure 2. Transposon targeting using a strategy based on 
protein-protein interactions between a targeting fusion pro-
tein and the SB transposase. (a) The targeting fusion protein 
consists of the tetracycline repressor (TetR) that binds to 
the TRE, a nuclear localization signal (NLS), a glycine-bridge 
(G) and the N-terminal protein interaction domain of the 
SB transposase (N-57). (b) Cells were cotransfected with 
the components of the transposon targeting system, and 
genomic DNA of pooled transformant cells was subjected 
to PCR as described in Fig. 5c. The agarose gel shows PCR 
products obtained from cells transfected with a vector ex-
pressing TetR/NLS/N-57 or with TetR/NLS/LexA with primers 
amplifying the left or the right IR of the transposon. M: size 
marker. (c) Mapping of targeted SB insertions (arrows) with 
respect to the TRE-EGFP target isolated from five inde-
pendent experiments is shown. Multiple arrows represent 
independent insertions into the same site. Positions of the 
insertions are indicated below; the numbers correspond to 
the base pair numbering of the pTRE-d2EGFP plasmid (Clon-
tech). (d) Frequency of targeted transposition. The agarose 
gel shows PCR products obtained from individual, trans-
genic cell clones, using primer pairs amplifying the right IR 
of the transposon. A PCR product recovered from pooled (P) 
DNA samples serves as a reference. M: size marker.

Figure 3. Bioenginered muscle implants

Figure 4. Compared to Mo-MuLV-LTR, Sleeping Beauty IRs 
exhibit moderate promoter activity. Luciferase-reporter 
experiments.

Figure 5. The expression of the neighbouring genes is influ-
enced primarily by the promoter activity of the transgene. 
The shielding effect of the SH4 insulator sequences influ-
ences. Luciferase-reporter experiments.

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New Therapies – Gene Therapy

12

gENE THERAPY

INTHER

INTHER  proposes  to  improve  the  performance 
of transposon vectors by enhancing their trans-
positional activity and by strengthening their bi-
osafety  profiles. The  project  intends  to  improve 
the performance of the SB and FP systems, to en-
sure  that  the  expressed  therapeutic  gene  does 
not  influence  the  genes  at  the  integration  site. 
It also aims to enhance the activity of the trans-
posase by in vitro evolution (DNA shuffling of hy-
peractive versions of the transposases).

The project will target SB/FP transposon integra-
tion into specific locations in the human genome 
in order to improve the safety profile of transpo-
son  vectors  for  gene  therapeutic  applications. 
Molecular  strategies  of  targeted  transposition 
employed  by  naturally  occurring  transposable 
elements will be adapted to the SB/FP systems. 

INTHER  seeks  to  establish  the  optimal  condi-
tions  to  deliver  the  recombinase  system/thera-
peutic  gene  combinations  for  the  different  cell 
types.  Since  the  transposon  is  a  plasmid  based 
non-viral delivery vehicle, cutting edge non-viral 
nucleic acid delivery methods will be utilised. In 
the project’s framework, Nucleofection technol-
ogy would be optimised specifically for each of 
the  recombinase  systems  in  the  context  of  the 
relevant  cell  types  targeted  in  this  project.  Si-
multaneously, the capabilities of cell-penetrating 
peptides  (CPPs),  in  combination  with  the  trans-
poson systems, will be evaluated. In addition, the 
application  of  reversible  implantation  systems 
(collagen implants, skin biopumps, and encapsu-
lated cells) will be explored.

A disease model system will be identified, where 
the recombinase technology has a clear advan-
tage over viral approaches and current non-viral 
approaches. To ascertain whether recombinase-
based  technologies  might  offer  an  alternative 
to  treatments  based  on  viral  transduction,  the 
recombinase  systems  will  be  probed  in  seven 
different ex vivo disease models, previously treat-
ed  by  viral  vectors.  The  selected  ex  vivo  model 

systems are well established, ‘single therapeutic 
gene’ models. Selective advantage of the treated 
cells  either  exists  or  can  be  provided,  using  the 
human  ABCG2  multidrug  transporter  protein 
as  a  selectable  marker  for  several  models.  The 
treatment of the selected diseases has not been 
solved by conventional treatments in these mod-
els, and they are currently established to use viral 
vectors. The animal disease models of INTHER in-
clude copper metabolism diseases, anaemia, hy-
percholesterolemia,  bleeding  disorders,  chronic 
granulomatous  disease  (CGD)  and  neurological 
disorders.

INTHER will address the interaction between the 
recombinase systems and the treated cell, in par-
ticular. Safety issues of transposon vector admin-
istration  and  the  characteristics  of  genomic  in-
tegration will also be tackled. Extensive datasets 
with  viral  vectors  are  available  in  these  models 
in order to evaluate the potential of the transpo-
son-based, non-viral approach.

Expected outcome:

INTHER is expected to establish new therapeutic 
tools for somatic gene therapy as an alternative 
to existing viral and non-viral technologies. The 
project  will  evaluate  the  potential  of  recombi-
nase-based  (Sleeping  Beauty,  Frog  Prince  and 
PhiC31-mediated)  vectors  for  safe  and  efficient 
transgene expression in ex vivo models. It will de-
velop  and  optimise  delivery  techniques  suiting 
the transposon technology. 

INTHER  is  investigating  seven  animal  disease 
models  to  establish  the  advantages  and  limita-
tions  of  the  recombinase-mediated  therapeutic 
approach. To address the problems of efficacy us-
ing non-viral technology, the experimental mod-
el  systems  are  carefully  selected.  In  most  of  the 
disease models, the treated cell population has a 
selective advantage. The use of selectable mark-
ers,  particularly  ABCG2,  is  a  promising  strategy 
that  will  enhance  the  selection  of  engineered 

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gENE THERAPY

cells. The experimental data will provide a strong 
basis for further validation of transposon-based 
vectors in preclinical and clinical settings. 

INTHER  expects  to  deliver  the  results  set  out 
below.

Main findings:

Breakthroughs: In the first year of the project, the 
proof of principle of manipulating the target site 
of integration was established.  

Major publications 

Liu,  J.,  Jeppesen,  I.,  Nielsen,  K.,  Jensen,  T.G.,  ‘Phi 
c31 integrase induces chromosomal aberrations 
in  primary  human  fibroblasts’,  Gene  Ther,  2006, 
Aug;13(15):1188-90.

Thorrez,  L.,  Vandenburgh,  H.,  Callewaert,  N., 
Mertens, N., Shansky, J., Wang, L., Arnout, J., Collen, 
D., Chuah, M., Vandendriessche, T., ‘Angiogenesis 
enhances factor IX delivery and persistence from 
retrievable  human  bioengineered  muscle  im-
plants’, Mol Ther, 2006, Sep;14(3):442-51.

• 
significantly more active recombinase 
systems  compared  to  the  prototype  ver-
sions,  to  ensure  the  feasibility  of  the  re-
combinase-based  gene  therapeutic  ap-
proach; 
•  proof  of  principle  experiments  tar-
geting  the  integration  sites  of  Sleeping 
Beauty and Frog Prince to predetermined 
genomic loci in human cells;
•  better  penetration  of  the  transposon 
into cells, through the use of cell-penetrat-
ing peptides (CPPs);
•  development of optimal protocols for 
the  combination  of  the  transposons  and 
Nucleofector® technology; 
•  utilising  transposon  vectors  and  ex 
vivo  therapeutic  approaches,  INTHER  will 
test the potential of clinically feasible and 
potentially  safer  approaches  for  the  fol-
lowing: somatic gene therapy of disorders 
in  copper  metabolism;  Biopump  laser 
beam gene transfer (BPLBGT) technology 
as a cell factory for the production of hu-
man  secretory  proteins;  gene  therapy  of 
the  genetic  bleeding  disorders  haemo-
philia  and  hereditary  thrombocytopenia; 
for  gene  therapy  of  CGD,  to  develop  a 
method to provide a selective advantage 
for modified stem cells, using the human 
ABCG2  multidrug  transporter  protein  as 
a  selectable  marker;  the  use  of  encapsu-
lated human cell lines and human neural 
progenitor  cells;  and  cell  therapy  in  LDL 
receptor-deficient mice; 
•  evaluation of safety and toxicity issues 
associated with the use of transposons as 
gene vectors in human cells.

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127

 
 
 
 
 
 
gENE THERAPY

PéterKrajcsi
Solvo Biotechnology, Inc.
Szeged, Hungary

LarsWahlberg
NsGene A/S 
Ballerup, Denmark

INTHER

Coordinator 

ZsuzsannaIzsvák
Max-Delbrück-Center for Molecular Medicine
Robert Rössle Strasse 10
13125 Berlin, Germany
E-mail: zizsvak@mdc-berlin.de

Partners

ÜloLangel
University of Stockholm
Department of Neurochemistry
Stockholm, Sweden

ThomasG.Jensen
The Kennedy Institute
National Eye Clinic 
Glostrup, Denmark

EithanGalun
Goldyne Savad Institute of Gene Therapy
Hadassah Medical Organisation
Jerusalem, Israel

SeppoYlä-Herttuala
University of Kuopio
A.I.Virtanen Institute
Department of Biotechnology and Molecular Medicine
Kuopio, Finland

MarineeChuah
Flanders Interuniversity Institute for Biotechnology vzw
University of Leuven 
Department of Transgene Technology and Gene Therapy 
Leuven, Belgium

BalázsSarkadi
National Medical Centre
Institute of Haematology and Immunology
Budapest, Hungary

HerbertMüller-Hartmann
AMAXA GmbH
Cologne, Germany

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gENE THERAPY

EPIvECTOR

Episomalvectorsasgenedeliverysystemsfortherapeuticapplication






ContractNo 
Projecttype

LSHB-CT-2004-1196
SpecificTargetedResearchProject

ECcontribution  e 2100000

Startingdate 1January200
Duration 36months

Website  www.ls.manchester.ac.uk/epivector

Background and objectives: 

There  are  many  chronic  human  diseases  that 
cause  great  suffering  as  a  result  of  inherited  or 
acquired mutations in our genetic material. Once 
present,  these  changes  to  the  structure  of  DNA 
are passed from one cell to the next as cells di-
vide.  Inherited  and  acquired  mutations  are  re-
sponsible for many diseases, common examples 
being cystic fibrosis and muscular dystrophy. In 
these  cases,  the  debilitating  consequences  of 
mutations  in  DNA  arise  because  of  changes  to 
the  structure  of  the  protein  that  is  expressed 
from the mutated gene. 

The concept of gene therapy is to overcome the 
damage caused by genetic mutations in human 
cells by providing, to the appropriate cells, a nor-
mal copy of the damaged gene. In principle, if the 
normal protein is then expressed in these target 
cells  it  will  be  possible  to  replace  the  malfunc-
tioning protein with a fully functioning counter-
part. However, this is technically very challenging, 
and  systems  that  are  currently  being  evaluated 
in clinical trials suffer from potential deficiencies 
that compromise safety. 

The  most  challenging  aspects  of  gene  therapy 
arise from two main sources. First, available pro-
tocols for delivering DNA to the target cells are 
generally  inefficient. The  most  efficient  systems 
use viral particles, but the best systems are also 
associated with immunological side effects. Sec-
ond,  even  when  the  DNA  is  delivered  to  target 

Figure 1: Distribution of pEPI-1 in CHO cells, during and 
immediately following mitosis. The localisation of the 
episome was studied by FISH on spreads of metaphase 
chromosomes (A), and the equal distribution of vector 
molecules was monitored in postmitotic nuclei of divid-
ing cells (B). The episome (green) was visualised by pEPI 
FISH. To-Pro-3 was used for DNA counterstaining (red). A 
maximum intensity projection was rendered from a set 
of 5 mid serial sections in (B). Arrows in (A) indicate a pEPI 
signal pair, each signal localised on a sister chromatid. 
Note the extremely high efficiency with which episomes 
are segregates to daughter nuclei - in (B) both daughters 
have 7 single plasmids defined by FISH signals. The FISH 
spots also show low grade mirror symmetry, similar to 
that seen for chromosome territories following mitosis.

cells, it is by no means certain that the gene will 
be  expressed  faithfully.  In  fact,  all  too  often  the 
protein  is  only  made  for  a  short  time.  One  way 
of providing long-term expression is to use vec-
tors  that  integrate  into  the  chromosome  of  the 
target cell; this ensures that the new gene is not 
lost as cells divide. However, a potential problem 
with  this  is  that  by  interfering  with  the  cell  ge-
nome, it is possible to alter the normal patterns 
of  gene  expression,  and  in  some  cases  this  can 
lead to cancer.

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129

 
 
 
EPIvECTOR

gENE THERAPY

Figure 2: Reporter gene expression in transgenic pig foetuses generated by sperm mediated gene transfer of epi-vectors. 
Shows an analysis of enhanced-green fluorescent protein expression in embryonic pig tissues after transfer of epi-vectors to 
oocytes using sperm mediated gene transfer. The episomal status of the epivectors was determined by blotting and plasmid 
rescue. Confocal images show reporter gene expression in six tissues (from left to right: skeletal muscle, heart, liver, kidney, 
lung, and skin) from a representative positive foetus  (Top) and the same tissues from a negative control foetus (Below).

This  project  is  designed  to  evaluate  the  possi-
bility  of  developing  extra-chromosomal  gene 
delivery systems for gene therapy and evaluate 
protocols for their safe use in pre-clinical model 
systems.  The  episomal  systems  under  study  do 
not interfere with host cell chromosomes and so 
do  not  have  any  secondary  genetic  effects. The 
key  features  of  EpiVector  are  to  define  the  ge-
netic  elements  that  are  required  for  persistent, 
long-term gene expression and maintenance of 
the  extra-chromosomal  DNA  molecules  in  ap-
propriate cells.

Approach and methodology: 

Key  approaches  are  to  use  vector  construction 
and  engineering  to  define  the  behaviour/prop-
erties  of  different  vector  constructs  in  different 
cellular  environments.  Models  systems  used  in-
clude: general cell culture models; model systems 
for gene therapy (e.g. muscular dystrophy); gene 
expression  in  primitive  haematopoietic  stem 
cells;  mouse  embryonic  stem  cells;  and  sperm 
mediated gene transfer to explore development 
behaviour.

The  key  feature  of  epi-vectors  is  their  excep-
tional  mitotic  and  expressional  stability  in  cells 

with  no  selection  pressure. The  main  limitation 
is  the  (low)  efficiency  with  which  highly  mitoti-
cally stable vectors are maintained. This appears 
to be a general property of extra-chromosomal 
DNA vectors. Establishing a stable maintenance 
phenotype  does  not  involve  genetic  changes 
(shown  by  plasmid  rescue  and  sequencing),  so 
it  is  clearly  dependent  on  epigenetic  program-
ming.  This  problem  has  been  studied  in  detail 
and  important  observations  are  beginning  to 
pinpoint the key features:

1. The Lipps and Jackson collaboration has 
shown that when stably maintained, vec-
tors  are  stably  associated  with  the  most 
active nuclear compartment and retain an 
association with some chromosomal land-
marks  throughout  mitosis.  This  provides 
a  means  of  ensuring  efficient  inclusion 
into  daughter  nuclei  but  also  –  though 
the molecular mechanism is unknown – a 
means of uniform segregation during cell 
division. 
2. The Lipps and Azorin collaboration has 
revealed  that  the  efficiency  of  establish-
ing  stable  clones  is  increased  5-10fold  if 
DNA  is  assembled  into  chromatin  before 
delivery to cells. 
3.  Bode  and  colleagues  have  shown  that 

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gENE THERAPY

generation of minicircles that do not con-
tain  bacterial  DNA  also  improved  estab-
lishment efficiencies by 5 to10 times.
4. Studies by Lipps and collaborators have 
shown that epi-vectors can be introduced 
into pig oocytes by sperm-mediated gene 
transfer to give an extremely high efficien-
cy of episomal gene transfer to all tissues 
of pig embryos. 
5. Establishment of human stem cell clones 
and mouse embryonic stem cell clones is 
ongoing.

Expected outcome: 

The  expected  outcome  of  the  project  is  to 
deliver a detailed understanding of the genetic 
and epigenetic features that support the stable 
mitotic behaviour of episomal DNA molecules in 
proliferating human cells. The project has defined 
the genetic elements required for stable mitotic 
inheritance  and  is  now  attempting  to  analyse 
the  epigenetic  features.  An  important  aspect 
of  this  activity  is  to  understand  in  molecular 
detail  how  epi-vectors  interact  with  functional 
nuclear  compartments  and  define  the  dynamic 
behaviour of these interactions. Live cell imaging 
of  tagged  epi-vectors  is  being  used  to  address 
vector dynamics. 

Main findings: 

Significant findings have included a phenotypic 
description  of  the  molecular  behaviour  of  epi-
vectors  in  proliferating  cells.  This  has  provided 
early  insight  into  the  molecular  behaviour  and 
provides  a  system  that  should  allow  the  mo-
lecular mechanism to be defined. It is clear that 
epigenetic programming of the vector is key to 
the way it behaves in proliferating cells. Modify-
ing  the  sequence  or  chromatin  architecture  of 
the  vector  prior  to  introducing  into  target  cells 
significantly influences the efficiency with which 
mitotically  stable  clones  are  established.  A  par-
ticular  breakthrough  in  this  regard  comes  from 

observations  made  using  sperm  mediate  trans-
fer of epi-vectors to pig oocytes. 

The oocyte is a permissive environment for epi-
genetic  re-programming  and  epi-vectors  have 
been shown to pass to all tissues of an embryo 
following  their  introduction  into  oocytes  by 
sperm mediated gene transfer. This implies that 
mouse embryonic stem cells will likely provide a 
route for making transgenic animals with a sta-
ble expression of genes from epi-vectors.  

Major publications 

Jenke,  A.C.W.,  Eisenberger,  T.,  Baiker,  A.,  Stehle, 
I.M., Wirth,  S.,  Lipps,  H.J., ‘The  nonviral  episomal 
replicating  vector  pEPI-1  allows  long-term  inhi-
bition  of  bcr-abl  expression  by  shRNA’,  Human 
Gene Therapy, 2005, 16:533-539.

Bode, J., Winkelmann, S., Götze, S., Spiker, S., Tsut-
sui,  K.,  Bi,  C.,  Benham,  C., ‘Correlations  between 
Scaffold/Matrix  Attachment  Region 
(S/MAR) 
Binding Activity and DNA Duplex Destabilisation 
Energy’,    J.  Mol.  Biol,  2006,    358:597-613.  http://
dx.doi.org/10.1016/j.jmb.2005.11.073

Jackson,  D.A.,  Juranek,  S.,  Lipps,  H.J.,  Design-
ing  nonviral  vectors  for  efficient  gene  transfer 
and long term gene expression’, Mol. Ther, 2006, 
14:613-626.

Winkelmann,  S.,  Klar,  M.,  Benham,  C.,  A.K.,  P., 
Goetze, S., Gluch, A., Bode, J., ‘The
positive aspects of stress: Strain Initiates Domain 
Decondensation  (SIDD)’,  Briefings  in  Functional 
Genomics  and  Proteomics,  2006,  5,  24-31  http://
dx.doi.org/10.1093/bfgp/ell003

Manzini, S., Vargiolu, A., Stehle, I., Bacci, M.L., Cer-
rito, M.G., Giovannoni, R., Forni, M., Donini, P., Papa, 
M., Lipps, H.J., Lavitrano, M., ‘Genetically modified 
pigs produced with a non vital episomal vector’, 
Proc. Natl, 2006, Acad. Sci. USA 103:17672-17677.

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gENE THERAPY

EPIvECTOR

Nehlsen, K., Broll, S., Bode, J., ‘Replicating minicir-
cles: Generation of nonviral episomes for the ef-
ficient  modification  of  dividing  cells’,  Gene  Ther 
and Mol Biol, 2006, 10B: 233-243.

Coordinator 

DeanAJackson
Manchester Interdisciplinary Biocentre
University of Manchester
Faculty of Life Sciences
131 Princess St
M1 7DN Manchester, UK.
E-mail: dean.jackson@manchester.ac.uk. 

Partners 

RoelvanDriel
University of Amsterdam
Science Faculty
Swammerdam Institute for Life Sciences
Amsterdam, Netherlands

HansLipps
University of Witten
Department of Cell Biology
Witten, Germany

GeorgeDickson
Royal Holloway - University of London
Centre for Biomolecular Sciences
Egham, UK

FernandoAzorin
CSIC Institute of Molecular Biology
Barcelona, Spain

JürgenBode
GBF
German Research Centre for Biotechnology
Braunschweig, Germany

AriellaOppenheim
Hebrew University-Hadassah Medical School
Department of Haematology
Jerusalem, Israel

BenDavis
genOway Germany
Hamburg, Germany

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gENE THERAPY

POLEXgENE

Biocompatible non-viral polymeric gene delivery systems for the exvivo
treatmentofocularandcardiovasculardiseaseswithhighunmetmedicalneed

ContractNo 
Projecttype

LSHB-CT-2006-019114
SpecificTargetedResearchProject

ECcontribution  e 2132607
Startingdate 1June2006

Duration 36months






Background and objectives:

PolExGene  aims  to  develop  a  non-viral  ex  vivo 
gene therapy that can find potential applications 
in the field of ocular and cardiovascular diseases 
with  high  unmet  medical  need.  One  of  the  key 
innovative aspects of PolExGene is the develop-
ment of novel gene vectors from non-toxic and 
non-immunogenic,  biodegradable  polymeric 
carrier materials based on multifunctional poly-
Δ-aminoacids.

Overall  DNA  delivery  will  be  improved  through 
the  combination  of  polyplexes  with  cell  pen-
etrating  peptides  (CPP). The  internalisation  effi-
ciency is expected to be enhanced through the 
use of Penetratin-like CPP, while the membrane-
cell interaction will be improved by functionalis-

ing the polymer membrane with cell interacting 
peptides (CIP).

Approach and methodology:

Two  different  strategies  will  be  developed  and 
compared. In a first strategy (A), cells will be trans-
fected  using  CPP  containing  polyplexes,  after 
which  the  cells  will  be  seeded  on  a  biodegrad-
able polymer membrane, functionalised with CIP. 
Polymer membranes will be prepared by solvent 
casting or electrospinning. 

Alternatively (strategy B), the polymer membrane 
will be surface coated with CPP-containing poly-
plexes prior to cell seeding. In both cases, the cell-

Figure 1. Application of an ex vivo gene therapy approach 
for the treatment of ocular and cardiovascular diseases.

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gENE THERAPY

POLEXgENE

seeded  biodegradable  polymer  membrane  will 
be implanted into the retina of the eye or used as 
coating for cardiovascular prosthesis. The biode-
gradable  polymers  selected  will  be  biocompat-
ible: non-toxic and non-immunogenic.

In  order  to  obtain  the  technological  objec-
tives,  the  following  scientific  objectives  will  be 
achieved by implementing seven Work Packages 
(WP): 

Coordinator

EtienneSchacht
Polymer Chemistry & Biomaterials Research Group
Department Organic Chemistry
Ghent University Krijgslaan 281 S-4
9000 Ghent, Belgium 
E-mail: etienne.schacht@ugent.be 

Partners

•  WP1: selection of CIP and CPP;
•  WP2:  development  of  CPP-containing 
polymers;
•  WP3:  development  of  CIP-containing 
polymer membranes; 
•  WP4: preparation of plasmids and CPP-
containing polyplexes; 
•  WP5:  characterisation  of  polyplex-cell 
and polymer membrane-cell interactions;
•  WP6:  study  on  immunological  prop-
erties  of  polyplexes  and  polymer  mem-
branes;
•  WP7:  polymer  membrane  implanta-
tion in test animals.

An eighth Work Package, WP8, will be devoted to 
the project management to ensure that the over-
all project objective is met.

The  technological  objectives  addressed  for  the 
development  of  CPP  containing  biocompatible 
non-viral polymeric gene delivery systems for ex 
vivo treatment are very ambitious: 

•  development  of  non-toxic  and  non-
immunogenic  biodegradable  polymeric 
CPP-containing DNA-carriers;
•  development of a biohybrid cell-based 
CIP-containing  implantation  system  for 
ocular and cardiovascular applications;
•  development of a cell-based micro ar-
ray  technology  for  the  quantification  of 
the  biological  activity  (transfection  effi-
ciency and toxicity) of the DNA-carriers. 

ArtoUrtti
University of Helsinki
Faculty of Pharmacy/Drug Discovery and Development 
Technology Center
Helsinki, Finland 

HagenThielecke
Fraunhofer Institute for Biomedical Engineering 
St. Ingbert, Germany

Prof.Blanca.Rihova
Institute of Microbiology
Prague, Czech Republic) 

AlainJoliot
Ecole Normale Supérieure
Paris, France

EberhartZrenner
University Eye Hospital of Tübingen 
Tübingen, Germany

AlainYvorra
Epytop 
Nîmes, France

DavidEckland
Ark Therapeutics
London, UK

SeppoYlä-Herttuala
University of Kuopio
A.I. Virtanen Institute 
Kuopio, Finland

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gENE THERAPY

MAgSELECTOFECTION

Combinedisolationandstablenonviraltransfectionofhaematopoieticcells–a
novelplatformtechnologyforex vivohaematopoieticstemcellgenetherapy

ContractNo 
Projecttype

LSHB-CT-2006-019038
SpecificTargetedResearchProject

ECcontribution  e 2800000
Startingdate 1May2006
Duration 36months






Website  www.magselectofection.eu

Background and objectives:

The feasibility of ex vivo gene therapy in humans 
has  been  demonstrated  with  retrovirally  trans-
duced  haematopoietic  stem  cells.  At  the  same 
time,  risks  associated  with  the  use  of  retroviral 
vectors  became  apparent.  In  order  to  circum-
vent  problems  associated  with  viral  vectors,  we 
will develop a novel, nonviral, combined ex vivo 
cell separation/transfection platform for haemat-
opoietic  cells,  suitable  for  site-specific  genomic 
integration of transfected nucleic acids into non-
coding regions of the host genome. This concept 
will be applied to haematopoietic stem cells and 
will  be  validated  with  established  preclinical 
models of SCID-X1.

This platform technology for integrated cell iso-
lation and transfection will be based on a clinical-
ly approved magnetic cell separation technique 
(MACS  Technology),  combined  with  magneti-
cally  enhanced  transfection  (Magnetofection). 
It  will  also  be  based  on  nucleic  acid  constructs 
that  provide  site-specific  genomic  integration 
— either the phage phiC31 integrase system or, 
alternatively, a drug-inducible AAV-derived repli-
case/integrase system.

Technology  validation  includes  the  following: 
analysis  of  genomic  integration  sites;  transcrip-
tom  profiling;  characterisation  of  stable  and  in-
ducible  trans-gene  expression,  and  evaluation 
of homing; and engraftment and persistence in 
transgenic  animal  models  using  molecular  bio-

logical tools, as well as magnetic resonance imag-
ing. The  therapeutic  potential  will  be  examined 
in  a  SCID-X  mouse  model  in  direct  comparison 
with  established  retroviral  technology.  Besides 
contributing to the progress of health care, Mag-
netoselectofection  will  foster  the  competitive-
ness of Europe’s biotechnology industry.

Magnetoselectofection  seeks  to  combine  mag-
netic  cell  sorting  and  transfection  based  on 
Miltenyi’s  clinically  approved  MACS Technology 
and  Magnetofection  (magnetically  guided  nu-
cleic acid delivery), for manipulation of haemat-
opoietic cells. It also aims to achieve a stable and 
regulatable transfected gene expression in hae-
matopoietic stem cells, by site-specific genomic 
integration  of  delivered  nucleic  acids  upon 
Magnetoselectofection, with plasmid constructs 
harbouring the phiC31 integrase system, and al-
ternatively,  a  drug-inducible  AAV-derived  repli-
case/integrase system.

Magnetoselectofection will characterise this tech-
nology through an analysis of genomic integration 
sites, transcriptom profiling, and a characterisation 
of stable and inducible transfected gene expres-
sion.  The  project  will  validate  the  technology  in 
transgenic  SCID-X  mouse  models  by  evaluating 
homing,  engraftment  and  persistence  in  trans-
genic  animal  models,  via  molecular  biological 
tools  and  magnetic  resonance  imaging.  Magne-
toselectofection will demonstrate the therapeutic 
efficacy, as well as assess the associated risks of the 
technology in transgenic mouse models.

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New Therapies – Gene Therapy

13

 
 
 
gENE THERAPY

MAgSELECTOFECTION

The  dissemination  and  use  of  Magnetoselecto-
fection  will  be  made  possible  from  its  transfer 
into research and clinics through the participat-
ing companies.

Approach and methodology:

The  clinical  success  in  curing  patients  suffering 
from SCID-X has demonstrated the great poten-
tial of gene therapy. In the same study, the short-
comings  and  serious  biological  risks  associated 
with the use of current viral gene vector technol-
ogy became evident. 

Of the 11 patients treated with retrovirally trans-
duced haematopoietic stem cells, 2 developed a 
lympho-proliferative  disease  due  to  insertional 
mutagenesis  caused  by  the  uncontrolled  inte-
gration properties of the vector. Hence, safe alter-
natives are required to realise gene therapeutic 
concepts  involving  stable  genetic  modification 
of cells.

Members of this consortium have previously de-
veloped  independent  technologies  for  the  fol-
lowing: magnetic cell separation; magnetic field-
assisted nucleic-acid delivery to cells; nucleic acid 
constructs  suitable  for  stable  integration  into 
eukaryotic genomes; disease-relevant transgenic 
mouse models, to evaluate the biological charac-
teristics and therapeutic potential of transgenic 
cells under pre-clinical settings; and tools for ex-
pression profiling. 

There are strong personal and scientific links to 
the EU-funded CONSERT project. The fundamen-
tal objective of Magselectofection is to combine 
the named independent technologies and skills 
so as to generate a novel, yet simple, efficient and 
safe integrated platform technology for the ge-
netic modification of cells in clinical and research 
applications.  This  innovative  technology  is  the 
first developed towards the application in SCID-
X1 gene therapy; clinical and research protocols 
are well established.

Expected outcome:

Magselectofection will generate a novel technol-
ogy for nucleic acid delivery which combines the 
ease  of  application  of  magnetic  nanoparticle-
based cell isolation and sorting, with the advan-
tages  of  magnetic  nanoparticle-based  nucleic 
acid  delivery  technology.  While  this  novel  plat-
form  technology  is  expected  to  be  broadly  ap-
plicable to a variety of clinical and research ap-
plications, this project focuses on its use in gene 
therapy  of  a  rare  hereditary  disease,  SCID-X1. 
Therefore, Magselectofection comprises detailed 
analyses  in  the  stable  genetic  modification  and 
transplantation  of  haematopoietic  stem  cells, 
while avoiding the use of viral gene vectors. 

Expected results include novel nucleic acid con-
structs  for  stable  genetic  modification  of  cells 
and novel technology for transfecting such con-
structs. In addition, they include insights into the 
molecular biology of such constructs once trans-
fected,  into  the  cell  biology  of  haematopoietic 
stem cells modified in this manner, and into the 
biodistribution, engraftment and homing of such 
cells once transplanted in animal models.

Apart from the primary intended application, i.e. 
gene therapy of SCID-X1, there are multiple nu-
cleic acid therapy applications that can be envis-
aged, as is indicated below.

•  using the method to induce RNA inter-
ference (RNAi) in hematopoietic stem cells 
to fight HIV infections;
•  using the method for the transfection 
of  lymphocytes  for  adoptive  immuno-
therapy of cancer;
• 
transfection of neuronal stem cells for 
ex  vivo  gene  therapy  of  central  nervous 
system disorders;
ex  vivo  transfection  of  stem  cells  or 
• 
progenitor  cells  for  tissue  engineering 
(bone,  cartilage  reconstruction,  tendon 
and wound healing etc.);
• 

cell  tracking,  molecular  imaging.  The 

136

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gENE THERAPY

PeterBabinec
Comenius University
Bratislava, Slovakia

OlivierZelphati
OZ Biosciences S.A.R.L.
Marseille, France

JosephRosenecker
Ludwig-Maximilians-Universität
Munich, Germany

UlfJohann
Fraunhofer Gesellschaft zur Förderung der angewandten 
Wissenschaft e.V.
Munich, Germany

MicheleCalos
Stanford University
Palo Alto (CA), USA

magnetic  nanoparticles  used  in  Magse-
lectofection  are  suitable  contrast  agents 
for magnetic resonance imaging. Thus, the 
method  can  be  used  to  introduce  these 
particles in any cell of interest, optionally 
along with other agents for molecular im-
aging (e.g. fluorescent dyes).

Coordinator 

ChristianPlank
Klinikum rechts der Isar der TU München
Institute of Experimental Oncology
Ismaninger Str. 22
D-81675 Munich, Germany
E-mail: plank@lrz.tum.de

Partners

GerardWagemaker
Erasmus Medical Centre
Rotterdam, Netherlands

FulvioMavilio
Fondazione Centro San Raffaele del Monte Tabor
Milan, Italy

TsveeLapidot
Weizmann Institute of Science
Rehovot, Israel

ZygmuntPojda
M. Sklodowska-Curie Memorial Cancer Centre and Insti-
tute of Oncology
Warsaw, Poland

MichaelApelandIanJohnston
Miltenyi Biotec GmbH
Bergisch-Gladbach, Germany

PeterSteinlein
Research Institute of Molecular Pathology
Vienna, Austria

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New Therapies – Gene Therapy

137

SYNTHEgENEdELIvERY

Ex vivogenedeliveryforstemcellsofclinicalinterestusingsyntheticprocesses
ofcellularandnuclearimportandtargetedchromosomalintegration

gENE THERAPY






ContractNo 
Projecttype

LHSB-CT-200-018716
SpecificTargetedResearchProject

ECcontribution  e 2400000

Startingdate 1December200

Duration 36months

Website  http://lepg.univ-tours.fr

Background and objectives:

The  project  will  develop  SyntheGeneDelivery 
(SGD),  a  new  ex  vivo  gene  delivery  (evGD)  pro-
tocol to provide stable long-term expression of 
integrated  transgenes.  The  clinical  objective  is 
to provide gene therapy solutions for some ge-
netic diseases of the neuromuscular and skeletal 
systems,  as  well  as  for  circulating  polypeptide 
deficiencies  that  together  afflict  more  than  35 
million patients in Europe.

In gene therapy for inherited disorders, the main 
area  of  application  of  an  efficient  evGD  proc-
ess lies in the modification of stem cells. Indeed, 
the  advantage  of  such  systems  is  that  cells  can 
be transfected and verified in vitro, before being 
grafted  in  a  patient  with  a  debilitating  genetic 
deficiency. 

Approach and methodology:

The main limitation in developing these therapeu-
tic strategies is the availability of efficient and safe 
processes. The SGD protocol is designed to over-
come  these  problems  and  to  ensure  long-term 
maintenance of transgene expression by chromo-
somal integration, bypassing several barriers: 

the  extra-cellular  matrix  and  the  cell 

the  traffic  from  endosomes  to  lyso-

• 
membrane; 
• 
somes and subsequent degradation; 
• 
• 

the dissociation of DNA from its carrier; 
the transfer into the nucleus; 

Murine mesenchymal stem cells transfected with 
plasmid DNA encoding GFP complexed with BGTC/DOPE 
liposomes.

the access to “good” target sites to en-
• 
sure healthy integrations in chromosomes; 
•  avoidance  of  undesirable  transgene 
integrations. 

To  achieve  these  goals  and  fully  develop  the 
technology,  SyntheGeneDelivery  will  use  two 
populations  of  stem  cells.  The  first  is  a  mesen-
chymal stem cell (MSC), which can be simply re-
covered from bone marrow and manipulated for 
the  correction  of  some  genetic  disorders,  such 
as  those  involving  haematopoietic  and  mesen-
chymal derivative cells. The second will be mus-
cle stem cells that have the potential to correct 
some muscle genetic disorders, but that can also 
be used, after the intramuscular graft of the mod-
ified stem cells, as a factory for the production of 
polypeptides  that  are  deficient  in  genetic  and 
acquired disorders, and that can be secreted by 
the muscle to diffuse toward some target organs 
of the patient. 

For  DNA  vector  internalisation  in  cells,  the  con-
sortium will use the BGTC-DOPE lipoplex system 

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gENE THERAPY

in the first version, and the Poloxamer block co-
polymer in the two latest versions. Transgene in-
tegration will be achieved using vectors derived 
from  the  mariner  Mos1  transposon. These  DNA 
vectors will be improved for their key properties 
using molecular engineering methods.

First, their integration efficiency will be improved, 
not only by optimising the vector nuclear import, 
but  also  by  obtaining  hyperactive  transposases 
(the enzyme assuming the transposon mobility), 
and ITR (the end of the transposon). Second, Syn-
theGeneDelivery will define the configuration of 
vectors  for  which  integration  efficiency  will  not 
be dependent on the transgene size. Finally, site-
specific  integration  vectors  will  be  developed 
by  fusing  mariner  Tpases  with  polydactyl  Zinc 
Finger Domains, thus targeting the DNA integra-
tion  at  close  proximity  of  a  predefined  site. The 
efficiency  and  the  fidelity  of  integration  of  the 
chimeric Tpases will be modelled in vivo before 
their use for clinical purposes.

At  each  step,  the  modifications  to  the  Synthe-
GenTransfer  process  will  be  evaluated  in  foetal 
and adult MSC, and in muscle stem cells. Their ef-
fect on retaining the dual stem cell properties on 
self renewal and multilineage differentiation will 
be evaluated, in comparison to standard lentivi-
ral integration systems. 

Expected outcome:

EvGD strategies for therapy purposes are of high 
interest  when  they  concern  stem  cells,  due  to 
the potential of differentiating them ex vivo and 
in vivo in a wide range of cell types. Indeed, this 
opens  key  possibilities  that  meet  challenges 
posed by many diseases. The SGD process is well 
adapted to develop evGD on stem cells because 
of the properties of the BGTC-DOPE, which is the 
only identified chemical product able to internal-
ise DNA molecules in these cells. Developing the 
project, the innocuity of the cellular import will 
be  improved  by  replacing  the  BGTC-DOPE  with 

The mariner Mos1 transposon.

the fully non-cytolytic product approved by the 
FDA — the Propoplex block copolymer — using 
procedures adapted to ex vivo transfection.

The consortium will use the SGD process on mus-
cle or mesenchymal stem cells, in an attempt to 
assay its therapeutic potential in several diseases: 
Duchenne Muscular Dystrophy (DMD), type 1 Os-
teogenesis imperfecta, Hemophilia A and Diabe-
tes mellitus type 1. The properties of SGD and its 
evolutions are adequate to the therapeutic strat-
egies targeted on these diseases. It can be used 
with cassettes containing the therapeutic trans-
gene that are less than 5 kpb for the DMD (using 
highly  engineered  microdystrophin  constructs), 
and less than 3-4 kbp for other diseases. Moreo-
ver, if mariner vector is able to carry larger trans-
genes, the consortium should then use cassettes 
containing therapeutic transgenes of 20-30 kpb.

Main findings:

Foetal  mesenchymal  stem  cells  and  a  clone  of 
murine  muscle  stem  cells  from  a  condition-
ally immortilised mouse have been selected for 
evaluation of all the SGD process. Human muscle 
stem cells will be used to evaluate the SGD V3.

DNA internalisation in stem cells is actually per-
formed  using  the  nucleofection  system  from 
Amaxa.  A  more  efficient  transfection  reagent,  a 
lipidic  aminoglycoside  derivative,  is  now  avail-
able from In Cell Art (Partners 2 and 7), and will 
be used to develop SGD V2.

MRNA encoding the transposase internalisation 
in HeLa cells is actually efficiently performed us-
ing PEI. The Kozak modified and codon optimised 
Mos1 transposase has little activity in transfected 

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New Therapies – Gene Therapy

139

gENE THERAPY

SYNTHEgENEdELIvERY

stem  cells.  A  second  version,  which  has  no  self-
annealing properties was developed by Partner 
1 and will be used in SGD V2. Hyper-active trans-
posases are still available and will be used in SGD 
V2.  Similarly,  more  efficient  ITR  were  designed 
and will be used in SGD V2.

In conclusion, it must be underlined that SGD V1 
was made from few optimised components (cel-
lular  import,  nuclear  import,  protein  and  DNA 
counterparts). Results were encouraging but not 
yet at the level expected at the end of the pro-
gramme. However, the necessary elements are in 
place, to proceed with evaluation of the SGD V2.

Major publications  

Sinzelle, L., Jegot, G., Brillet, B., Rouleux-Bonnnin, 
F., Bigot, Y., Augé-Gouillou, C., ‘Factors controlling 
Mos1 transposition in vitro and in bacteria’, Sub-
mitted.

Svahn,  M.G.,  Hasan,  M.,  Sigot,  V.,  Valle-Delgado, 
J.J., Rutland, M.W., Lundin, K.E., Smith, C.I., ‘Self-as-
sembling supra-molecular complexes by single-
stranded extension from plasmid DNA’, Oligonu-
cleotides, 2007, Spring;17(1):80-94.

Ge, R., Heinonen, J.E., Svahn, M.G., Mohamed, A.J., 
Lundin, K.E., Smith, C.I., ‘Zorro locked nucleic acid 
induces sequence-specific gene silencing’, FASEB 
J, 2007, Apr 10.

Coordinator 

YvesBigot
Université François Rabelais de Tours
LEPG FRE 2969
UFR Sciences & techniques 
37200 Tours, France
E-mail: bigot@univ-tours.fr 

Partners 

CyrilleGrandjean
In Cell Art
Nantes, France

OscarSimmonson
Avaris
Stockholm, Sweden

KarinLundin
Karolinska Institutet
Stockholm, Sweden

RonaldChalmers
University of Oxford
Oxford, UK

DominicWells
Imperial College
London, UK

BrunoPitard
INSERM
Nantes, France

140

New Therapies – Gene Therapy

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11/8/07   10:49:32 AM

gENE THERAPY

MOLEdA

Molecularoptimisationoflaser/electrotransferDNAadministrationintomuscle
andskinforgenetherapy






ContractNo 
Projecttype

LHSB-CT-2004-12034
SpecificTargetedResearchProject

ECcontribution  e 2447972

Startingdate 1January200
Duration 36months

Website  www.moleda.org

Background and objectives:

For gene therapy, the use of non-viral DNA offers 
the  advantage  of  lack  of  immunogenicity,  ab-
sence of size limit for the therapeutic expression 
cassette, simpler GMP production, and improved 
safety/toxicity  profiles.  However,  the  efficient, 
precise  and  safe  delivery  of  plasmids  or  other 
forms of non-viral DNA remains to be improved.

Two different in vivo local tissue plasmid delivery 
techniques  were  recently  introduced,  and  are 
currently the most efficient in terms of gene ex-
pression level: electrotransfer using mild electric 
pulses  (ET)  and  laser  beam  gene  transfer  using 
femtosecond infrared titanium sapphire laser en-
ergy (LBGT).

Approach and methodology:

MOLEDA  aims  to  determine  the  optimal  condi-
tions  for  precise  and  selective  plasmid  transfer 
into  skeletal  muscle  and  skin,  in  the  frame  of  a 
head-to-head  comparison  between  the  two 
most  promising  in  vivo  plasmid  DNA  delivery 
technologies at present, and to introduce molec-
ular strategies to improve these technologies. 
Optimised  conditions  will  then  be  used  on  ani-
mals with four different therapeutic applications. 
The overall objective is to develop non-viral DNA 
technology into a preclinical phase. Consequent-
ly, MOLEDA will address safety issues (tissue dam-
age, inflammation, etc.) to ensure the success of 
its efforts.

Moleda’s Team - Brussels

More precisely, MOLEDA aims at identifying the 
optimal  conditions  for  plasmid  delivery  into 
muscle  and  skin.  This  optimisation  will  be  ac-
companied  by  a  head-to-head  comparison,  us-
ing the same DNA preparations, between ET and 
LBGT. This optimisation/comparison will be per-
formed in two different tissues:  muscle and skin. 
MOLEDA will, for each tissue and each model of 
delivery  (ET  or  LBGT),  assess  which  is  the  best 
promoter: cytomegalovirus (CMB) or two differ-
ent  tissue-specific  promoters.  For  muscle,  two 
strong skeletal muscle promoters will be exam-
ined.  For  skin  vaccination,  one  strongly  active 
keratinocyte  promoter,  and  one  dendritic  cell 
specific promoter will be used.
The researchers will assess and select the prefer-
ential form of non-viral DNA to be used, from the 
following options: 

conventional plasmid; 

• 
•  prokaryotic-backbone  deleted,  ‘mini-
plasmid’  devoid  of  antibiotic  resistance 

NewTherapy_02.indd   141

11/8/07   10:49:37 AM

New Therapies – Gene Therapy

141

 
 
 
 
 
MOLEdA

gENE THERAPY

gene (an approach pioneered by Team 1); 
linear  PCR  produced  eukaryotic  ex-
• 
pression  cassette  (technology  mastered 
by Team 2).

These  optimisation  results  will  be  applied  to 
three  different  therapeutic  paradigms,  for  four 
different medical applications: 

Moleda’s big team

MOLEDA will assess, for the secreted transgenic 
protein part of the programme, the usefulness of 
an optimised enhancing secretion discovered re-
cently (Tauler et al., 1999), as well as examine for 
each tissue (muscle and skin), and for each mode 
of delivery (ET and LBGT), what the best DNA for-
mulation  is. The  options  are ‘naked’  versus  self-
associated  to  cationic  lipid,  or  to  non  cationic 
lipid (discovered by Team 1; Patent Herscovici et 
al. 2002). To date, cationic lipids have proved in-
efficient for naked DNA intramuscular injection, 
but except for that particular case, little is known 
about the effect of cationic lipids, and even less 
of the effect that neutral DNA formulation has on 
ET and on LBGT in different tissues.

The  researchers  will  study  if  pre-  or  post-ionto-
phoresis may be beneficial for ET or LBGT. Ionto-
phoreisis involves applying low intensity electri-
cal currents, <0,5mA/cm2, for several minutes. It 
has  been  shown  to  enhance  transdermal  drug 
delivery  and  promote  intratissular  migration  of 
charged molecules.

long-term  intracellular  expression  of 
• 
the dystrophin gene in skeletal muscle for 
the  therapy  of  Duchenne  muscular  dys-
trophy  (an  inherited  neuromuscular  dis-
ease); 
long-term  blood  secretion  of  a  circu-
• 
lating  protein,  with  erythropoietin  (EPO) 
and  secreted  monoclonal  antibodies  as 
the selected applications; 
• 
short-term  transgene  expression  in 
skin  for  raising  humoral  and  cellular  im-
mune  response:  antitumour  vaccination 
(i.m. ET has been shown to enhance DNA 
vaccine response).

It  is  likely  that  each  specific  therapeutic  appli-
cation  will  require  an  adapted  protocol  of  DNA 
administration. Internal data among the consor-
tium’s  participants  confirm  this  assumption.  For 
instance,  electrotransfer  might  represent  the 
best technology for skeletal muscle DNA delivery 
when  maximal  production  and  blood  secretion 
of the transgenic protein is needed. Converesely, 
LBGT  might  represent  a  more  efficient  technol-
ogy for skin administration or for the production 

142

New Therapies – Gene Therapy

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gENE THERAPY

Coordinator 

DanielScherman
U640 Inserm, UMR 8151 CNRS
Faculté de Sciences Pharmaceutiques et Biologiques,
4 avenue de l’Observatoire
75270 Paris Cedex 06, France
E-mail: daniel.scherman@univ-paris5.fr

Partners

EithanGalun
Hadassah Medical Organisation
Jerusalem, Israel

VéroniquePréat
Université Catholique de Louvain
Louvain, Belgium

GeorgeDickson
Royal Holloway & Bedford New College
University of London
London, UK

VolkerSchirrmacher
Deutsches Krebsforschungszentrum
Heidelberg, Germany

IacobMathiesen
INOVIO
Oslo, Norway

PaulParren
GENMAB
Utrecht, Netherlands

StéphaneBlot
Ecole Nationale Vétérinaire d’Alfort
Maison-Alfort, France

AlainCimino
Inserm Transfert
Paris, France

by muscle of a very potent hormone. Also, a mus-
cle specific promoter might prove more adaptive 
(i.e.  leading  to  high  level  sustained  transgene 
expression),  only  to  ET  or  LBGT.  In  addition,  the 
precision of gene delivery might depend on the 
delivery technology used.

Expected outcome:

Optimised conditions and constructs for ET and 
LBGT will be applied in the context of the three 
different  gene  therapy  paradigms,  and  four  dif-
ferent medical applications. The safety issues (tis-
sue damage, inflammation, integration, etc.) will 
be specifically addressed as well. The overall ob-
jective  is  to  develop  non-viral  DNA  technology 
into a preclinical phase.

Main findings:

In the first part of the programme, ET and LBGT 
were optimised in parallel by local DNA delivery 
into  skeletal  muscle  and  skin,  by  assessing  the 
following: 

•  what the best promoter (ubiquitous or 
tissue-specific) is for each model of deliv-
ery (ET or LBGT); 
• 
the preferential form of non-viral DNA 
—  either  conventional  plasmid  or  linear 
PCR produced expression cassette;
the usefulness of an optimised secre-
• 
tion  sequence  for  secreted  transgenic 
proteins; 
•  what  the  best  DNA  formulation  is  — 
‘naked’ versus associated to cationic or to 
non-cationic lipid; 
• 

if pre- or post-iontophoresis is beneficial.

In addition, MOLEDA has made great progress by 
constructing a new generation of high efficiency 
plasmids  devoid  of  antibiotic  resistance  genes 
and  their  corresponding  genetically  modified 
bacterial host.

NewTherapy_02.indd   143

11/8/07   10:49:40 AM

New Therapies – Gene Therapy

143

 
 
 
 
 
ANgIOSKIN

DNAelectrotransferofplasmidscodingforantiangiogenicfactorsasaproofof
principleofnon-viralgenetherapyforthetreatmentofskindisease.

gENE THERAPY

ContractNo 
Projecttype

LSHB-CT-200-12127
SpecificTargetedResearchProject

ECcontribution  e 2780683
Startingdate 1May200
Duration 48months






Website 

http://dl.ltfe.org/login_angioskin.asp

Background and objectives:

The  ANGIOSKIN  consortium  wants  to  establish 
the proof of concept that therapeutic genes can 
be safely delivered to skin by DNA electrotrans-
fer  (electrogenetherapy),  in  order  to  prevent  or 
to treat acquired or inherited skin diseases. 

The ANGIOSKIN project is based on the results of 
the earlier CLINIPORATOR (Analysis of the mech-
anisms of DNA electrotransfer, and elaboration of 
a CE labelled pulse generator), and ESOPE (Prep-
aration  of  the  Standard  Operating  Procedures 
of  Electrogenetherapy,  electrotransferring  of  a 
reporter  gene  into  humans)  projects  and  on  a 
proprietary gene coding for a potent human an-
tiangiogenic factor. This factor is the disintegrin 
fragment  of  the  Metargidin,  also  referred  to  as 
AMD-15 (MDC 15 in mouse).  

trodes  for  safety  and  efficacy  on  normal 
skin in animals, and analyse the effects of 
the electrotransfer of the antiangiogenet-
ic factor on models of skin disease related 
to excessive angiogenesis, using non non-
vasive  biophysical,  as  well  as  histological 
methods to follow changes in the vascu-
larisation of the lesion.
•  electrotransfer  the  therapeutic  gene 
to  a  benign  lesion  in  humans,  as  a  proof 
of  concept  of  the  use  of  non-viral  gene 
therapy to treat acquired or inherited skin 
diseases.

Final goal:

ANGIOSKIN  will  bring  the  proof  of  concept  of 
therapeutic  gene  non-viral  electrotransfer  into 
skin, to treat inherited or acquired diseases.

Approach and methodology:

Main findings:

ANGIOSKIN will do the following:

•  electrotransfer  the  proprietary  thera-
peutic antiangiogenesis gene to melano-
ma  cutaneous  metastasis  in  humans,  us-
ing the procedures validated in the ESOPE 
project,  in  order  to  show  its  clinical  ef-
ficacy.  Noninvasive  biophysical  methods 
(Doppler ultrasounds, Near InfraRed Spec-
troscopy, etc.) will be used to monitor the 
antiangiogenic effects.
•  develop  new  specific  electrodes  for 
skin  lesions  treatment,  validate  the  elec-

The  consortium  has  been  actively  working  on 
the gene therapy vector that will be used in the 
clinical  trials  foreseen  in  the  project. The  issues 
related to the GMP production of the DNA for the 
clinical  trials  have  also  been  cleared  with  sub-
contractors. The sequences required for gene ex-
pression (promoters) have also been worked out, 
resulting  in  the  latest  data  showing  improved 
expression and efficacy of the vector. 

The preparation of the clinical trials has started. 
The clinical partners involved in the first clinical 

144

New Therapies – Gene Therapy

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11/8/07   10:49:40 AM

 
 
 
 
 
 
 
trials  (related  to  malignant  tissues,  i.e.  melano-
mas)  have  defined  the  protocols.  Despite  the 
delay  in  the  submission  of  the  protocols  to  the 
ethics  committees  and  regulatory  agencies,  the 
preparation  of  these  documents  and  contacts 
has continued. 

The technological aspects have resulted in proto-
types for new devices. The extent of their future 
use  is  still  under  evaluation  by  the  consortium. 
Moreover, other items are also under experimen-
tal  evaluation  for  eventual  inclusion  in  future 
patent applications. Portions of the work of the 
consortium will soon be presented in specialised 
meetings. 

Major publications

Patents:

Two patent applications have been prepared and 
submitted. They cover some of the microfluidics 
aspects of the project, as well as the pulses de-
livered by the new type of electrodes on which 
the  consortium  is  working.  However,  their  sum-
maries have yet to be published and no details 
can be provided at the moment. 

Mir,  L.M.,  Miklavcic,  D., ‘Combinations  of  electric 
pulses for DNA electrotransfer’.

Jullien,  M.C.,  Hoel,  A.,  Mir,  L.M., ‘Fluid  dispensing 
system’.

gENE THERAPY

Coordinator 

LluisM.Mir
UMR 8121 CNRS - Institut Gustave-Roussy
Laboratory of Vectorology and Gene Transfer 
39, rue C. Desmoulins
F- 94805 Villejuif Cédex, France
E-mail: luismir@igr.fr 

Partners

GonzaloCabodevila
Franche Comté Electronique Mécanique Thermique et 
Optique
Besançon, France

DamienGrenierandCarolineJullien
CNRS UMR 8029 SATIE (Systèmes et Applications de 
Technologies de l’Information et de l’Energie) Team 
BIOMIS (biomicrosystèmes)
ENS Cachan Campus de Kerlann
Bruz, France

RuggeroCadossi
IGEA S.r.l.
Carpi (MO), Italy

CarolineRobert
Institut Gustave-Roussy
Dermatology Department of Medicine
Villejuif Cédex, France

VéroniquePréat
Université catholique de Louvain
Unité de Pharmacie galénique
Brussels, Belgium

JulieGeh
Herlev Hospital
Deptarment of Oncology, 54B1
Herlev, Denmark

NewTherapy_02.indd   145

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New Therapies – Gene Therapy

14

gENE THERAPY

ANgIOSKIN

MichaelP.Schön
University of Würzburg
Rudolf Virchow Zentrum für Experimentelle Biomedizin
Würzburg, Germany

DominiqueCostantini
BioAlliance Pharma SA
Paris, France

DamijanMiklavcic
University of Ljubljana
Faculty of Electrical Engineering
Ljubljana, Slovenia
Subcontractor SC2 to partner University of Ljubljana:

GregorSersa
Institute of Oncology
Laboratory of Radiation Biology
Ljubljana, Slovenia

LoneSkov
Afdelingslæge
Dermatologisk afdeling
KAS Gentofte
Hellerup, Denmark

146

New Therapies – Gene Therapy

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11/8/07   10:49:40 AM

TOTAL
participants
%
nb
0,6
1
0,6
1
1,2
2
0,6
1
1,2
2
1,2
2
0,6
1

GVPNConference
4-5 October 2001
EVRY
TOTAL
participants
%

nb

Country

EUROLABCOURSE
14-27 April 2002
EVRY

CONFERENCE & COURSE
1-14 February 2004
 BELLATERRA

CONFERENCE & COURSE
14-26 June 2004
EVRY

TOTAL
participants
nb
%

Participants in the
practical course

nb

%

TOTAL
participants
nb
nb

Participants in the
practical course

gENE THERAPY

Participants in the
practical course

nb

nb

%

%

2

2

4

3

1

1

1

1



8
2

3
1

1,40

12,5

6,25

2,10

0,36

0,70

3,13

3,13

1
1
1

1
10

1
13

42
17

1
2
2
1
1

223
8

25,0
6,25

0,36
4,63

0,70
7,10

1,97
0,66

79,36
2,85

30,00
12,10

45
3
2
5
1
1

3,13
3,13
3,13

0,66
1,32
1,32
0,66
0,66

29,61
1,97
1,32
3,29
0,66
0,66

ContractNo 
Projecttype

ECcontribution  e 16100

1
64
8
LSSB-CT-2003-03219
1
6
SpecificSupportAction
1
3
3
14
1
Website  www.vecteurotrain.org

INvIvOvECTORTRAIN

1.   Algeria
2.   Austria
3.   Belgium
4.   Brazil
5.   Bulgaria
6.   Canada
7.   China
European labcourse: towards clinical gene therapy: preclinical gene transfer
8.   Czech Republic
9.   Denmark
assessment
10.  Finland
11.  France
12.  Germany
13.  Greece
14.  Hungary
15.  Iceland
16.  Iran
17.  Ireland
18.  Israel
19.  Italy
20. Japan
21.  Korea
22.  Latvia
23.  Lithuania
24.  Luxembourg
25.  Mexico
26.  Netherlands
27.  Poland
28.  Portugal
29.  Romania
30.  Russia
31.  Slovenia
32.  Spain
33.  Sweden
34.  Switzerland
35.  Turkey
36.  Taiwan
37.  United Kingdom
38.  Uruguay
39.  USA

Background and objectives:
1,78
0,71
1,07

Startingdate 1June2003
3,13
1,97
6,25
Duration 21months
1,32
0,66

1
Main findings:
6
1

3,13
3,13
6,25
15,63
3,13

0,6
0,6
6,0
1,8
3,6
4,2
0,6
6,0

3,13
12,5
3,13
3,13
3,13

0,6
38,3
4,8
0,6
3,6

0,66
1,97
0,66
2,63

5,00
7,90
3,60
0,70

0,70
0,70
1,40
0,70

21,88
12,5
3,13

0,6
1,8
1,8
8,4
0,6

3,13
3,13
6,25


1,42
1,42


3,13
9,38
3,13

3,29
0,66
3,95

18,75
3,13

46
3
3
2

7
11
5
1

3,0
0,6
1,2

3,13
3,13

3,13
3,13

0,70
0,70

3,13
9,38

1,40
4,30

1
4
1
1
1

1
1
2
5
1

1
3
1
4

1
1
2
1

5
1
2

1
1
2

6,25

3,13

1,32

6,25

3,13

1,07

4,30

0,66

3,13

9,38

12,5

3,13

6,25

3,13

5,00

1
3
1

3
2
1

7
4
1

5
1
6

5
2
3

4,8

1
1

1
1

1
1

1
3

1
2

2
6

4
4

12

2

1

2

2

3

6

1

1

4

3

1

1

2

7

2

32

12

281

100

100

100

140

0,71

4,27

8,60

Total

22 countries
44 % women

The  InVivoVectorTrain  event,  sponsored  by  the 
EC, was the third in a series of training initiatives 
on  gene  and  cell  therapy  that  Généthon  devel-
oped, with the participation of other centres ac-
tive in this field in the EU. Launched in 2001 with 
the  extensive  GVPN  conference  (Evry  of  France, 
13 countries
45 % women
4-5 October), the InVivoVector Train project con-
131  institutions
tinued with three workshops: the Eurolabcourse 
(105 academic, 22 
industries, 4 other
on vectorology in Evry, 14-26 April 2002, and two 
organisations(1))
in 2004 in Bellaterra (Spain, this report) and again 
in Evry, 14-26 June . This educational activity ad-
EC-MC-FP5, AFM, INSERM,
EC-MC-FP5, AFM,
dresses a primary need of the scientific commu-
GENOPOLE, EMBO, Industries
Industries
nity to update scientific concepts and technolo-
gies on gene therapy, in order to implement its 
developmental  process  towards  a  new  medical 
practice. 

87 institutions
(53 academic, 29 industries, 5 other
organisations)

patient’s associations, ethical/regulatory agencies, scientific press

13 countries
75 % women

(1)

Scheme of the training events in 2002 and 2004 (see 
www.vecteurotrain.org for more information)

32

32

152

100

100

100

100

30,26
1,97
1,97
1,32

18 countries
65 % women

29 countries
39 % women

18 countries
50 % women

29 countries
49.5  % women

73 institutions
(58 academic, 13 industries, 2 other 
organisations)

1
1
10
3
About 150 researchers hailing from different in-
6
7
stitutions across the EU and abroad participated 
1
10
in the last three events.  The success of the confer-
ence and course in Bellaterra was also due to the 
8
167
new  training  format,  that  combined  the  typical 
research  conference  (including  invited  lectures, 
short communications and poster sessions) with 
a  round  table  discussion  and  a  practical  course 
(see the table in the annex). After the basic tech-
niques  of  vector  production,  purification  and 
characterisation  were  delivered  with  the  first 
S P O N S O R S
Eurolabcourse in 2002 in Evry, the InVivoVector-
Train consortium thought it necessary to provide 
training on the basic techniques of gene transfer 
in vivo  into  animals,  which  is  the  second  essen-
tial step in gene therapy development. Therefore, 
lectures,  short  communications  and  posters  on 
the principles of gene delivery into different or-
gans  (including  gene  expression  assessment  in 

101  institutions
(74 academic, 25 industries, 2 other 
organisations)

EC-LSH-FP6, GENOPOLE, CG-
Essonne, INSERM, Industries

EC-LSH-FP6, FEBS, INSERM,
GENOPOLE, Industries

T O T A L    n u m b e r   o f    i n s t i t u t i o n s :    2 9 3 ( 2 2 6   a c a d e m i c   a n d   6 7   i n d u s t r i e s ) 

Deliverables:

Concepts

Tutorials
Lectures, discussions 

International keynote
speakers

Activities:

Teachers:

Participants:

4 days
   Conference

Examples

Research Data
Posters, short 
communications

Researchers

Good practices (1),
business opportunities

Public round table
Discussions with large public
exhibits, video-shows

Industries, regulatory agencies,
patients’ associations, opinion groups

Participants from all institutions(2)

32  selected young researchers(3)

8 days

  Practical course

Experience

Practical training

Experts/ teachers/tutors

(1) GLP, GMP, GCP, ethics, regulations... (2) 200 max PhD students, post docs, research staff, industries, regulators, other professionals (3)
PhD students, post docs, young researchers, selected on the basis of scientific excellence and motivation to participate

NewTherapy_02.indd   147

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New Therapies – Gene Therapy

147

 
 
 
INvIvOvECTORTRAIN

gENE THERAPY

Country

1.   Algeria
2.   Austria
3.   Belgium
4.   Brazil
5.   Bulgaria
6.   Canada
7.   China
8.   Czech Republic
9.   Denmark
10.  Finland
11.  France
12.  Germany
13.  Greece
14.  Hungary
15.  Iceland
16.  Iran
17.  Ireland
18.  Israel
19.  Italy
20. Japan
21.  Korea
22.  Latvia
23.  Lithuania
24.  Luxembourg
25.  Mexico
26.  Netherlands
27.  Poland
28.  Portugal
29.  Romania
30.  Russia
31.  Slovenia
32.  Spain
33.  Sweden
34.  Switzerland
35.  Turkey
36.  Taiwan
37.  United Kingdom
38.  Uruguay
39.  USA

GVPNConference
4-5 October 2001
EVRY
TOTAL
participants
%

nb

1
13

0,36
4,63

1

0,36

223
8

79,36
2,85

4
4

3

5
2
3

12

2

1,42
1,42

1,07

1,78
0,71
1,07

4,27

0,71

100

EUROLABCOURSE
14-27 April 2002
EVRY

CONFERENCE & COURSE
1-14 February 2004
 BELLATERRA

CONFERENCE & COURSE
14-26 June 2004
EVRY

TOTAL
participants
nb
%

Participants in the
practical course

nb

%

TOTAL
participants
nb
nb

Participants in the
practical course

%

nb

1
10

1

3

42
17

2

2
6

1
1
2
1

6

1
1

7
11
5
1

12

7

140

0,70
7,10

0,70

2,10

30,00
12,10

1,40

1,40
4,30

0,70
0,70
1,40
0,70

4,30

0,70
0,70

5,00
7,90
3,60
0,70

8,60

5,00

100

3
1

1
2
2
1
1

45
3
2
5
1
1

3
2
1

2

1
3
1
4

1

46
3
3
2

5
1
6

8
2

2

1
2

1

1

1
1

7
4
1

1

25,0
6,25

6,25

3,13
6,25

3,13

3,13

3,13
3,13

21,88
12,5
3,13

3,13

32

100

152

1,97
0,66

0,66
1,32
1,32
0,66
0,66

29,61
1,97
1,32
3,29
0,66
0,66

1,97
1,32
0,66

1,32

0,66
1,97
0,66
2,63

0,66

30,26
1,97
1,97
1,32

3,29
0,66
3,95

100

1
1
1

4

1
3
1

2

2

1
1
2

1

6
1

3

1

3,13
3,13
3,13

12,5

3,13
9,38
3,13

6,25

6,25

3,13
3,13
6,25

3,13

18,75
3,13

9,38

3,13

TOTAL
participants
%
nb
0,6
1
0,6
1
1,2
2
0,6
1
1,2
2
1,2
2
0,6
1

1
64
8
1
6

1
3
3
14
1

5
1
2

1
1
10
3
6
7
1
10

8

0,6
38,3
4,8
0,6
3,6

0,6
1,8
1,8
8,4
0,6

3,0
0,6
1,2

0,6
0,6
6,0
1,8
3,6
4,2
0,6
6,0

4,8

100

Participants in the
practical course

nb

%

1

1

1
4
1
1
1

1
1
2
5
1

1
1

4

1
3

2

32

3,13

3,13

3,13
12,5
3,13
3,13
3,13

3,13
3,13
6,25
15,63
3,13

3,13
3,13

12,5

3,13
9,38

6,25

100

Total

281

32

100

167

13 countries
45 % women

131  institutions
(105 academic, 22 
industries, 4 other
organisations(1))

22 countries
44 % women

13 countries
75 % women

29 countries
49.5  % women

18 countries
50 % women

29 countries
39 % women

18 countries
65 % women

87 institutions
(53 academic, 29 industries, 5 other
organisations)

73 institutions
(58 academic, 13 industries, 2 other 
organisations)

101  institutions
(74 academic, 25 industries, 2 other 
organisations)

T O T A L    n u m b e r   o f    i n s t i t u t i o n s :    2 9 3 ( 2 2 6   a c a d e m i c   a n d   6 7   i n d u s t r i e s ) 

EC-MC-FP5, AFM,
Industries

EC-MC-FP5, AFM, INSERM,
GENOPOLE, EMBO, Industries

EC-LSH-FP6, FEBS, INSERM,
GENOPOLE, Industries

EC-LSH-FP6, GENOPOLE, CG-
Essonne, INSERM, Industries

S P O N S O R S

(1)

patient’s associations, ethical/regulatory agencies, scientific press

Statistics on the participants in the training events on 
gene therapy since 2001

148

New Therapies – Gene Therapy

NewTherapy_02.indd   148

Deliverables:

Activities:

Concepts

Tutorials

Lectures, discussions 

Teachers:

International keynote

speakers

Participants:

4 days
   Conference

Examples

Research Data

Posters, short 

communications

Researchers

Good practices (1),

business opportunities

Public round table

Discussions with large public

exhibits, video-shows

Industries, regulatory agencies,

patients’ associations, opinion groups

Participants from all institutions(2)

32  selected young researchers(3)

11/8/07   10:50:00 AM

8 days

  Practical course

Experience

Practical training

Experts/ teachers/tutors

(1) GLP, GMP, GCP, ethics, regulations... (2) 200 max PhD students, post docs, research staff, industries, regulators, other professionals (3)

PhD students, post docs, young researchers, selected on the basis of scientific excellence and motivation to participate

gENE THERAPY

Coordinator 

MauroMezzina
Généthon-CNRS FRE 3018
1bis Rue de l’International
91002 Evry, France
E-mail: mezzina@genethon.fr

live  animals  and  the  immune  response),  were 
followed by a practical training on gene deliver-
ing in vivo, i.e. administration into liver, lung, brain 
and muscle.

In  this  course,  preclinical  gene  transfer,  and  the 
theoretical  and  practical  issues  of  gene  vectors 
as potential new bio-pharmaceuticals were pre-
sented. For the first time, 32 researchers from 18 
countries  were  trained  on  the  techniques  of  in 
vivo gene transfer, based on the criteria of good 
laboratory practices and the ethical principles of 
animal experimentation.

Major publications 

Mezzina, M., ‘Towards clinical gene therapy: pre-
clinical  gene  transfer  assessment’,  Gene Therapy, 
2004, vol. 11, S1 pp 1-172

NewTherapy_02.indd   149

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New Therapies – Gene Therapy

149

INdUSTRYvECTORTRAIN

Europeanlabcourse:advancedmethodsforindustrialproduction,purification
andcharacterisationofgenevectors

gENE THERAPY






ContractNo 
Projecttype

LSSB-CT-2003-016
SpecificSupportAction

ECcontribution  e 172000

Startingdate 1January2004
Duration 21months

Website  www.vecteurotrain.org

Background and objectives:

Approach and methodology:

The  topics  developed  in  IndustryVectorTrain 
focused  on  the  biotechnological  issues  of  gene 
therapy  (GT)  products  (vectors,  plasmids,  cell 
lines,  etc.),  and  when  they  must  be  prepared  at 
pharmaceutical  scale.  It  is  a  mandatory  step  in 
the development process of gene therapy. A to-
tal of 114 scientific communications (36 full lec-
tures, 23 short communications and 55 posters) 
were presented at a conference, highlighting the 
biology,  use,  and  industrial  development  of  the 
main gene vectors with general and expert infor-
mation on the following: 

some  applications  in  gene  transfer 

•  RVV,  LVV,  AdV  and  parvovirus-based, 
non-viral  and  other  viral  (alphavirus  and 
herpes-based) vector systems; 
• 
into liver, muscle, CNS and HSC; 
•  biotechnological  issues  of  scaling  up, 
i.e.  theory  and  praxis  of  purification,  cell 
cultures  techniques  and  devices  for  in-
dustrial  production  of  RTV,  AdV  and  AAV 
vectors, as well as quality control analysis; 
• 
the  regulatory  and  ethical  issues  of 
gene transfer and GT; 
• 
in  a  special  session  or  round  table  ,  a 
number  of  vector  production  facilities 
and  services  currently  available  to  the 
European  and  US  scientific  communities 
were presented. 

During the practical course, 32 selected research-
ers  acquired  hands-on  experience  in  advanced 
methods for the following: 

the  production  of  AAV  (i.e.  the  tran-
• 
sient transfection of 293 cells or infection 
of  insect  cells  with  recombinant  baculo-
viruses) and purification with simple and 
fast  methods  using  new  ion-exchange 
chromatographic devices; 
• 
large  batches 
the  preparation  of 
of  AdV  purified  with  chromatographic 
procedures; 
the  production  of  LVV  vectors  at  a 
• 
large scale with improved packaging cell 
lines; 
•  new  strategies  to  purify  RVV  and  LVV 
pseudotyped with VSV-G protein; 
• 
the characterisation of viral vectors for 
basic  bio-safety  parameters  (i.e.  physical 
and  functional  titration  and  assessment 
of adventitious contaminants and RCPs). 

Main findings:

Researchers  and  other  GT  professionals  attend-
ing  this  course  were  trained  in  the  scientific, 
technological  and  regulatory  issues  concerning 
the evolutionary process of gene therapy prod-
ucts towards new biopharmaceuticals. This could 
be  achieved  by  integrating  the  skills  of  differ-
ent professions: the researchers, who devise the 
gene transfer prototypes and protocols, and per-

10

New Therapies – Gene Therapy

NewTherapy_02.indd   150

11/8/07   10:50:01 AM

 
 
 
 
 
 
 
 
 
 
 
 
 
gENE THERAPY

Coordinator 

MauroMezzina
Généthon-CNRS FRE 3018
1bis Rue de l’International 
91002 Evry, France
E-mail: mezzina@genethon.fr

form animal experimentation to validate proofs 
of principle; the regulators, who define the legal 
frame  where  gene  therapy  products  must  be 
used in clinical trials; the industrialists, who allow 
technology transfers from the research benches 
to the biotech factories in order to transform the 
prototypes  into  exploitable  manufactures;  and 
the  physicians,  who  are  deeply  involved  in  the 
last steps which lead to the completion of a clini-
cal trial. 

Major publications 

Bagnis,  C.,  Merten,  O.W.,  Mezzina,  M., ‘Advanced 
methods  for  industrial  production  purification 
and characterisation of gene vectors’, Gene Ther-
apy, 2005, vol. 12, S1 pp 1-176

NewTherapy_02.indd   151

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New Therapies – Gene Therapy

11

12

New Therapies – Immunotherapy and Transplantation

NewTherapy_02.indd   152

11/8/07   10:50:02 AM

NewTherapy_02.indd   153

11/8/07   10:50:02 AM

IMMUNOTHERAPY ANd TRANSPLANTATION

14

New Therapies – Immunotherapy and Transplantation

NewTherapy_02.indd   154

11/8/07   10:50:03 AM

IMMUNOTHERAPY ANd TRANSPLANTATION
ALLOSTEM

Thedevelopmentofimmunotherapeuticstrategiestotreathaematologicaland
neoplasticdiseasesonthebasisofoptimisedallogenicstemcelltransplantation






ContractNo 
Projecttype

LHSB-CT-2004-03319
IntegratedProject

ECcontribution  e 8000000
Startingdate 1April2004

Duration 48months

Website  www.allostem.org

Background and objectives: 

Diseases of the blood, be they malignant or not, 
are  often  terminal.  Over  the  last  30  years,  im-
provements  in  chemotherapy  and  the  use  of 
external agents in cancer treatments have been 
progressing  steadily.  Despite  these  advances, 
however,  haematopoietic  stem  cell  transplanta-
tion remains the only therapy that can result in 
long-term disease-free survival of many patients, 
especially  those  who  have  had  relapses  follow-
ing initial sessions of chemotherapy. 

It is also now possible to approach the treatment 
of these diseases through the exploitation of the 
genetic  differences  between  individuals,  to  pro-
duce targets through which the immune system 
can  eliminate  the  diseased  cells.  This  treatment, 
immunotherapy, involves the stem cell transplant 
delivering specific immune effector cells expand-
ed from the donor. The scientific and technologi-
cal  objectives  of  AlloStem  are  to  develop  new 
strategies  for  optimising  the  use  of  allogeneic 
haematopoietic  stem  cell  transplantation,  based 
on the prediction and modulation of the immune 
response in order to achieve the following: 

reduce the risk of graft versus host dis-

• 
ease (GvHD); 
•  generate  selective  anti-tumour 
mune responses; 
•  provide  protective  immunity  against 
opportunistic infections; 
•  extend  the  applicability  of  transplan-
tation to a larger population of patients. 

im-

AlloStem  is  developing  protocols  for  the  treat-
ment  of  patients  with  haematological  disease, 
and for the effective delivery of immunotherapy 
to those patients. 

Approach and methodology: 

The AlloStem consortium has brought together 
29 clinical, research and industrial partners from 
the  EU  and  abroad. The  24  biomedical  research 
teams  and  5  SMEs  involved  as  full  partners  are 
devoted to the advance of translational research 
and new technology for the development of im-
munotherapeutic  strategies  to  treat  haemato-
logical and neoplastic diseases. In line with their 
objectives, the consortium’s research efforts are 
organised  in  three  main  work  packages  (WPs), 
wherein the different participating groups coop-
erate and constitute integrated task forces (TFs) 
to address the following topics:

•  development  of  immunotherapeutic 
strategies to mediate anti-tumour activity; 
the application of immunotherapeutic 
• 
strategies  to  control  infectious  diseases 
which threaten the lives of transplant re-
cipients; 
the optimisation of the transplant pro-
• 
cedure as a basis for subsequent immuno-
therapy.

NewTherapy_02.indd   155

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New Therapies – Immunotherapy and Transplantation

1

 
 
 
 
 
 
 
 
 
 
IMMUNOTHERAPY ANd TRANSPLANTATION

ALLOSTEM

Expected outcome: 

AlloStem targets improved healthcare for EU citi-
zens through new treatment protocols and phar-
maceuticals. As a result of the AlloStem training 
programme,  knowledge  in  this  important  field 
will be broadened and developed in those coun-
tries involved. 

Main findings: 

The results for the first 33 months have been im-
pressive across each work package. The tumour 
immunotherapy  work  package  (WP1)  has  char-
acterised  several  new  potentially  immunogenic 
peptides.  Algorithms  have  been  developed  to 
allow  for  the  determination  of  new  potential 
targets  of  immunotherapy.  Several  important 
signals  from  toll-like  receptors  were  identified, 
marking these pathways in dendritic cells as po-
tential targets, thus allowing manipulation of the 
induction of a cellular immune response.

selecting  and  enriching  antigen  spe-

Protocols have been developed for the purposes 
indicated below:
• 
cific T cells;
•  allowing  the  modification  of  malig-
nant cell populations, which result in the 
generation of malignant antigen present-
ing cells;
selecting and enriching antigen specif-
• 
ic T cells following the activation by these 
cell  populations,  using  cytokine  capture 
reagents; 
conducting  important  studies  to  bet-
• 
ter understand the interaction of Natural 
Killer (NK) cells with target tumour cells.

HLA class I and class II ligands of HCMV, EBV and 
other  relevant  infectious  pathogens. These  new 
ligands  have  been  incorporated  into  assays  for 
the  monitoring  of  virus-specific  immune  recon-
stitution  following  allogeneic  HSCT,  as  well  as 
in  protocols  of  T  cell  stimulation  and  priming, 
to generate virus-specific T cell lines and clones 
for adoptive immunotherapy. Expression of toll-
like receptors on dendritic cells has been studied 
after  stimulation  with  various  infectious  patho-
gens. Protocols were developed for the selection 
of  pathogen-specific T  cells  using  the  cytokine-
capture  assay.  Standard  operating  procedures 
were  developed  for  the  generation  of  clinical 
grade CMV- and EBV-specific T cell lines. 

The  transplant  modalities  work  package  (WP3) 
has  defined  new  conditioning  regimens  of  re-
duced  toxicity  and  sufficient  immuno-suppres-
sive  qualities  that  allows  more  therapeutically 
targeted transplants of haematopoietic progeni-
tors  from  HLA  mismatched  donors,  using  differ-
ent strategies of cell manipulation that enhance 
the contributions of regulatory T cells, veto cells, 
mesenchymal  stem  cells  or  NK  cells.  Protocols 
have been developed for the following purposes:
transplants  of  umbilical  cord  blood 

• 
supported by third party cells; 
• 
cell selection and cell purging and/or 
enrichment, using methods based on the 
immune  recognition  of  specific  cell  sur-
face or functional markers.

Standard  operating  procedures  for  post-trans-
plant assessment of tolerance (both in terms of 
rejection and of GvHD), as well as for the recov-
ery  of  protective  immune  functions  were  also 
defined. Clinical assays based on these protocols 
were initiated.

It should be noted that a number of crucial steps 
have  also  been  taken  to  ensure  new  immuno-
therapeutic strategies in the near future. 

Major publications

The infectious disease work package (WP2) has 
achieved the prediction and verification of new 

Ghiringhelli, F., Puig, P.E., Roux, S., Parcellier, A., Sch-
mitt, E., Solary, E., Kroemer, G., Martin, F., Chauffert, 
B., Zitvogel, L., ‘Tumor cells convert immature my-

16

New Therapies – Immunotherapy and Transplantation

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IMMUNOTHERAPY ANd TRANSPLANTATION

eloid dendritic cells into TGF-beta secretors that 
stimulate the proliferation of CD4+CD25+ regu-
latory  T  cells’,    Journal  of  Experimental  Medicine, 
2005, 202:919-929 (Epub Sept 26).

Marcenaro,  E.,  Della  Chiesa,  M.,  Bellora,  F.,  Paro-
lini,  S.,  Millo,  R.,  Moretta,  L.,  Moretta,  A., ‘IL12  or 
IL4  prime  human  Natural  Killer  cells  to  mediate 
functionally divergent interactions with dendrit-
ic  cells  or  tumors’,  Journal  of  Immunology,  2005; 
174:3992-3

Orabona, C., Puccetti, P., Vacca, C., Bicciato, S., Lu-
chini,  A.,  Fallarino,  F.,  Bianchi,  R.,  Velardi,  E.,  Per-
ruccio,  K.,  Velardi,  A.,  Bronte,  V.,  Fioretti,  M.C., 
Grohmann,  U.,  ‘Towards  the  identification  of  a 
tolerogenic  signacells’,  Blood,  2005;  Dec  8  (Epub 
ahead of print).

Taieb, J, Chaput, N, Ménard, C, Apetoh, L, Péquignot, 
M, Casares, N, Terme, M, Flament, C, Maruyama, K, 
Opolon, P, Lecluse, Y, Métivier, D, Tomasello, E, Vivi-
er, E, Ghiringhelli, F, Martin, F, Klatzmann, D, Poy-
nard, T, Yagita,  H,  Ryffel,  B,  Kroemer,  G.,  Zitvogel, 
L., ‘A  novel  dendritic  cell  subset  involved  in  tu-
mor immunosurveillance’, Nature Medicine, 2006, 
12:214-219 (Epub Jan 29).

Stewart,  C.A.,  Laugier-Anfossi,  F.,  Vely,  F.,  Saul-
quin,  X.,  Riedmuller,  J.,  Tisserant,  A.,  Gauthier,  L., 
Romagne,  F.,  Ferracci,  G.,  Arosa,  F.A.,  Moretta,  A., 
Sun, P.D., Ugolini, S., Vivier, E., ‘Recognition of pep-
tide-MHC  class  I  complexes  by  activating  killer 
immunoglobulin-like  receptors’,  Proceedings Na-
tional Academy of Sciences, USA, 2005, 102:13224-
13229.

Shaw,  B.,  Marsh,  S.G.E.,  Mayor,  N.P.,  Russell,  N.H., 
Madrigal,  J.A.,  ‘HLA-DPB1  matching  status  has 
significant  implications  for  recipients  of  unre-
lated  donor  stem  cell  transplants’,  Blood,  2006, 
107:1220-1226.

Einsele,  H.,  Reusser,  P.,  Bornhäuser,  M.,  Kalhs,  P., 
Ehninger,  G.,  Hebart,  H.,  Chalandon,  Y.,  Kröger, 

N.,  Hertenstein,  B.,  Rohde,  F.,  ‘Oral  valganciclo-
vir leads to higher exposure to ganciclovir than 
intravenous ganciclovir in patients following al-
logeneic  stem  cell  transplantation’,  Blood,  2006, 
107: 3002-3008.

Coordinator

AlejandroMadrigal
The Anthony Nolan Trust
The Royal Free Hospital
Pond Street
Hampstead, London NW3 2QG, UK.
E-mail a.madrigal@medsch.ucl.ac.uk 

Partners 

AlloStemLtd
London, UK

LeonidAlexeev
State Research Centre Institute of Immunology
Moscow, Russia

JirinaBartunkova
Univerzita Karlova V Praze
Prague, Czech Republic

JavierBordone
ITMO Fundacion Mainetti
Buenos Aires, Argentina

DominiqueCharron
HLA et Médecine 
Paris, France

Hans-GeorgRammensee
Eberhard Karls-Universitaet
Tuebingen, Germany

FredFalkenburg
University of Leiden Medical Centre
Leiden, Netherlands

New Therapies – Immunotherapy and Transplantation

17

NewTherapy_02.indd   157

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ALLOSTEM

IMMUNOTHERAPY ANd TRANSPLANTATION

ManuelFernandez
Universidad Autonoma de Madrid
Madrid, Spain

JuanGarcia
Centre de Transfusio I Banc de Teixits 
Barcelona, Spain

ElsGoulmy
University of Leiden Medical Centre
Leiden, Netherlands

NikolaiSchwabe
Proimmune Ltd
Oxford, UK

RobertRees
Nottingham Trent University
Nottingham, UK

YairReisner
Weizmann Institute of Science
Rehovot, Israel

FrançoisRomagne
Innate Pharma
Marseille, France

DoloresSchendel
GSF Forschungszentrum fuer Umwelt und Gesundheit 
GmbH
Munich, Germany

AndreaVelardi
Università Degli Studi di Perugia
Perugia, Italy

EricVivier
Centre National de la Recherche Scientifique
Marseille, France

LaurenceZitvogel
Institut Gustave Roussy
Villejuif Cedex, France

HermannEinsele
Julius-Maximilians-University
Wuerzburg, Germany

OysteinAamellem
Dynal Biotech ASA
Oslo, Norway

AndrzejLange
Ludwik Hirszfeld Institute
Wroclaw, Poland

FrancoLocatelli
IRCCS Policlinico San Matteo
Pavia, Italy

MarioAssenmacher
Miltenyi Biotec GmbH
Bergisch Gladbach, Germany

AlessandroMoretta
Università degli studi di Genova
Genoa, Italy

LorenzoMoretta
Istituto Giannina Gaslini
Genoa, Italy

RicardoPasquini
Universidade Federal do Parana
Parana, Brazil

PavelPisa
Karolinska Institutet
Stockholm, Sweden

18

New Therapies – Immunotherapy and Transplantation

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IMMUNOTHERAPY ANd TRANSPLANTATION
dC-THERA

Dendriticcellsfornovelimmunotherapies






LHSB-CT-2004-12074
ContractNo 
Projecttype NetworkofExcellence

ECcontribution  e 7600000

Startingdate 1January200
Duration 60months

Website  www.dc-thera.org

Background and objectives: 

Dendritic  cell  (DC)  immunobiology  has  enor-
mous potential for the development of new im-
munotherapies for cancer and infectious disease. 
Europe  is  home  to  a  critical  mass  of  leaders  in 
the  field,  who  have  pioneered  many  advances 
and  provided  initial  proof  of  principle  for  the 
approach. DC-THERA is a Network of Excellence 
(NoE) whose goal is to promote the integration 
of  the  activities  of  26  participant  groups  of  sci-
entists  and  clinicians,  along  with  6  expert  SMEs 
across Europe, over its five-year duration. 

DC-THERA  will  incorporate  additional  groups, 
particularly  from  new  and  candidate  Member 
States,  as  Associated  Members  of  the  Network. 
Their  collective  expertise  and  resources  will  be 
forged into an ambitious joint programme of ac-
tivities, so as to restructure the field. The project 
will  translate  genomic,  proteomic  and  bioinfor-
matic  information,  with  knowledge  from  mo-
lecular  cell  biology  and  preclinical  models,  into 
therapeutic endpoints: clinical trials of DC-based 
therapies for cancer and HIV. 

Approach and methodology: 

Four  thematic  S&T  Clusters  have  been  defined, 
with  a  fifth  for  horizontal  activities.  The  latter 
includes  development  of  synergistic  links  with 
other  networks,  providing  added  value  to  EC 
programmes  by  underpinning  all  projects  de-
veloping  new  vaccine  strategies  for  major  killer 

Fig 1. Langerhans cells (a type of dendritic cell) and 
gamma-delta T cells in skin epidermis

diseases. The network will provide a centralised 
European resource of databases for the field. 

DC-THERA will develop new research tools, inte-
grate existing and new technological platforms, 
recruit additional support staff, and make these 
available as shared resources for all partners. It will 
implement an ambitious education and training 
programme,  including  new  PhD  studentships,  a 
visiting scholars scheme, and high-quality train-
ing courses, with the possibility of a postgradu-
ate  degree  in  translational  DC  immunobiology. 
DC-THERA  will  contribute  to  the  European  bio-
technology  sector  and  have  a  major  impact  on 
European policymaking for the future.

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and MRI, has been used to visualise intracellular 
signalling  events,  intercellular  communication 
in  real  time,  and  cell  migration  in  living  tissues. 
In  WP5,  intracellular  signalling  pathways  have 
been investigated in different subsets of DC, and 
during their responses to different stimuli. Small 
inhibitory RNA (siRNA) constructs have been pre-
pared and delivered to DC to explore the effect 
of  interfering  with  these  pathways.  In WP6,  the 
responses of DC to ‘intrinsic’ stimuli (e.g. inflam-
matory  cytokines,  CD40  ligation)  and  ‘extrinsic’ 
stimuli (e.g. toll-like receptor, TLR, agonists) have 
been studied in depth, and some of the findings 
will facilitate the generation of more potent im-
munostimulatory DC for clinical use. 

Cluster 3,  Global  Responses,  comprises  two 
WPs: DC as adjuvants in vivo (WP7) and pre-clini-
cal studies (WP8). In WP7, two approaches have 
been  taken  to  enhance  immune  responses  in-
duced  by  DC,  either  by  targeting  particular  re-
ceptors  on  DC  themselves,  or  by  targeting  dif-
ferent cell types, such as natural killer (NK) cells, 
NKT  cells  and  gamma-delta  T  cells,  to  exploit 
their bidirectional interactions with DCs. In WP8, 
a  variety  of  preclinical  models  of  infectious  dis-
ease and cancer have been used, respectively, to 
investigate  the  role  of  DCs  in  immunity  against 
viruses (LCMV and HSV-1, the latter in neonates) 
and  bacteria  (Mycobacterium  tuberculosis  and 
listeria),  as  well  as  to  explore  various  strategies 
to  enhance  anti-tumour  responses  that  include 
depletion of regulatory T cells. 

Cluster 4,  Therapeutic  Applications,  comprises 
three WPs: clinical trials (WP9), immunomonitor-
ing (WP10) and regulatory affairs (WP11).  In WP9, 
clinical  trials  of  DC-based  immunotherapy  for  a 
variety of cancers including chronic lymphocytic 
leukaemia,  melanoma  and  renal  cell  carcinoma 
are  continuing,  while  others  are  in  advanced 
stages of planning, and a retrospective critical re-
view of published trials will be undertaken in the 
next  period.  In WP10,  a  critical  review  of  immu-
nomonitoring approaches has also been under-

Fig 2. Scanning electron micrograph of dendritic cells (e.g. 
the cell with ‘veils’ at the right hand side) interacting with 
resting T cells and an activated T cell (small round cells 
and large round cell, respectively)

Expected outcome: 

DC-THERA will evolve into a centre of excellence 
for DC Biology, with a lasting and global impact.  

Main findings: 

By  the  end  of  its  second  year,  the  network  has 
made  considerable  progress  in  all  areas.  Some 
highlights are as follows: 
Cluster 1,  Genes  and  Proteins,  comprises  three 
work packages (WPs): genomics (WP1), proteom-
ics (WP2) and bioinformatics (WP3). In WP1, tran-
scriptional profiling of DCs exposed to different 
microbial stimuli has resulted in the observation 
that surprisingly similar patterns of gene expres-
sion may be involved in cellular responses to dif-
ferent agents. In WP2, initial proteomic mapping 
of subcellular organelles of DC, including phago-
somes and exosomes, is in progress. And in WP3, 
testing of a database that will ultimately link ge-
nomic and proteomic information is under way. 

Cluster2, Molecular Cell Biology, also comprises 
three WPs:  advanced  imaging  (WP4),  intracellu-
lar signalling (WP5) and in vitro activation of DC 
(WP6).  In WP4,  a  variety  of  imaging  techniques, 
including confocal and two photon microscopy, 

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taken, and initial consideration has been given to 
the future standardisation of immunomonitoring 
approaches. Finally, in WP11, standard operating 
procedures  for  DC  generation  and  quality  con-
trol, and templates for clinical trial protocols and 
case report forms have been prepared. These will 
be made available to different centres as models 
before the preparation of master standard oper-
ating procedures and templates commences, in 
the next period. 

the 

infrastructure  underpinning 

As  an  NoE,  DC-THERA  seeks  to  enhance  not 
only  the  integration  of  joint  research  activities, 
as  exemplified  by  the  four  S&T  clusters,  but 
also 
the 
fields  of  DC  immunobiology,  vaccinology  and 
immunotherapy  in  Europe.  Cluster  5  has  been 
structured to meet the latter goal, and it comprises 
four WPs. The first, ‘promoting integration’ (WP12), 
is designed to enhance the research capabilities of 
the network, and to remove obstacles hampering 
progress,  by  establishing  core  technological 
platforms  and  promoting  the  development  of 
shared tools and protocols. The second, ‘ensuring 
excellence’ (WP13), focuses on the development 
of  education  and  training  activities,  the  sharing 
and  dissemination  of  expertise  and  resources 
with  groups  from  outside  of  the  network,  and 
the  forging  of  collaborative  links  with  other 
networks. 

Three  technological  platforms  have  now  been 
established, as part of WP12, for genomics, imag-
ing and cell therapeutics in order to enhance the 
research infrastructure for S&T clusters 1, 2 and 4, 
respectively. For example, the genomics platform 
has provided microarray analyses as a service to 
other  partners  who  would  not  necessarily  nor-
mally  have  access  to  such  specialised  expertise 
and resources. The platforms are also responsible, 
as part of WP13, for delivering the training activi-
ties  for  the  network,  which  include  theoretical 
courses, practical workshops and individual train-
ing  for  specific  purposes.  To  date,  the  network 
has provided a total of six high quality courses or 

Fig 3. Magnetic resonance (MR) imaging  of lymph node 
region before and after DC vaccination. The patient was 
injected with superparamagnetic iron oxide(SPIO)-la-
beled DC in a lymph node of the inguinal lymph node 
region under ultrasound guidance. MR imaging of this 
region was performed before (fig 1a,c,e) and 48 hours  
after (fig 1b,d,f ) the injection of the DC. Arrows indicate 
lymph nodes that show a decreased signal intensity after 
vaccination indicating the presence of SPIO-DC.

workshops on gene expression profiling and bio-
informatics: DC migration and dynamic imaging 
techniques; good manufacturing practice (GMP) 
and good clinical practice (GCP). 

As  part  of  its  education  activities  in  WP13,  DC-
THERA  has  created  13  new  PhD  positions  for 
young  researchers  who  target  collaborative  re-
search projects between 21 of the network’s 32 
partners. These positions are fincanced for a total 
of 37 person years. It has also organised an annual 
Graduate School for Young Investigators, the first 
of which was held in Celerina, St Moritz of Swit-
zerland, in March 2006. The remaining activities 
in WP13 include an associated partner scheme in 

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dC-THERA

which specific groups from outside the network 
are  invited  to  participate  in  its  activities  for  the 
mutual  benefit  of  both,  and  for  the  exploration 
of potential collaborative interactions with other 
networks in related fields. 

To date, a total of ~40 additional groups and SMEs 
working in the area have been integrated as as-
sociated  partners,  including  several  from  new 
and  candidate  member  states.  Moreover,  close 
interactions  with  a  STREP  in  the  area,  DC-VACC, 
has resulted in two joint meetings between the 
networks. WP13 also deals with communications 
issues and includes the development of the net-
work website at www.dc-thera.org, which is now 
integrated  with  a  new  management  and  com-
munications  software  tool  for  all  partners  and 
associated  partners.  The  remaining  two  WPs  of 
Cluster  5, ‘exploiting  results’  (WP14)  and ‘main-
taining  infrastructures’  (WP15)  deal  with  man-
agement  issues  per  se  and  look  forward  to  the 
future sustainability and continued impact of the 
network after the end of the contract. 

Major publications 

Savina, A., Jancic, C., Hugues, S., Guermonprez, P., 
Vargas, P., Moura, I.C., Lennon-Dumenil, A.M., Sea-
bra, M.C., Raposo, G., Amigorena, S., ‘NOX2 controls 
phagosomal  pH  to  regulate  antigen  processing 
during crosspresentation by dendritic cells’, Cell, 
2006, Jul 14;126(1):205-18. 

Koch,  M.,  Stronge,  V.S.,  Shepherd,  D.,  Gadola, 
S.D., Mathew, B., Ritter, G., Fersht, A.R., Besra, G.S., 
Schmidt, R.R., Jones, E.Y., Cerundolo, V., ‘The crys-
tal  structure  of  human  CD1d  with  and  without 
alpha-galactosylceramide’,  Nat  Immunol,  2005, 
Aug;6(8):819-26.

De  Vries,  I.J.,  Lesterhuis,  W.J.,  Barentsz,  J.O.,  Ver-
dijk,  P.,  van  Krieken,  J.H.,  Boerman,  O.C.,  Oyen, 
W.J., Bonenkamp, J.J., Boezeman, J.B., Adema, G.J., 
Bulte,  J.W.,  Scheenen, T.W.,  Punt,  C.J.,  Heerschap, 
A.,  Figdor,  C.G., ‘Magnetic  resonance  tracking  of 

dendritic  cells  in  melanoma  patients  for  moni-
toring  of  cellular  therapy’,  Nat  Biotechnol,  2005, 
Nov;23(11):1407-13. 

Van Hall, T., Wolpert, E.Z., van Veelen, P., Laban, S., 
van der Veer, M., Roseboom, M., Bres, S., Grufman, 
P.,  de  Ru,  A.,  Meiring,  H.,  de  Jong,  A.,  Franken,  K., 
Teixeira,  A.,  Valentijn,  R.,  Drijfhout,  J.W.,  Koning, 
F.,  Camps,  M.,  Ossendorp,  F.,  Karre,  K.,  Ljunggren, 
H.G.,  Melief,  C.J.,  Offringa,  R., ‘Selective  cytotoxic 
T-lymphocyte  targeting  of  tumor  immune  es-
cape variants’, Nat Med, 2006, Apr;12(4):417-24. 

Sporri,  R.,  Reis  e  Sousa,  C., ‘Inflammatory  media-
tors  are  insufficient  for  full  dendritic  cell  activa-
tion and promote expansion of CD4+ T cell pop-
ulations  lacking  helper  function’,  Nat  Immunol, 
2005, Feb;6(2):163-70. 

Frentsch, M., Arbach, O., Kirchhoff, D., Moewes, B., 
Worm, M., Rothe, M., Scheffold, A., Thiel, A., ‘Direct 
access  to  CD4+ T  cells  specific  for  defined  anti-
gens  according  to  CD154  expression’,  Nat  Med, 
2005, Oct;11(10):1118-24. 

Napolitani,  G.,  Rinaldi,  A.,  Bertoni,  F.,  Sallusto, 
F.,  Lanzavecchia,  A.,  ‘Selected  Toll-like  receptor 
agonist  combinations  synergistically  trigger  a 
T  helper  type  1-polarizing  program  in  dendritic 
cells’, Nat Immunol, 2005, Aug;6(8):769-76.

Taieb, J., Chaput, N., Menard, C., Apetoh, L., Ullrich, 
E., Bonmort, M., Pequignot, M., Casares, N., Terme, 
M.,  Flament,  C.,  Opolon,  P.,  Lecluse,  Y.,  Metivier, 
D., Tomasello, E., Vivier, E., Ghiringhelli, F., Martin, 
F., Klatzmann, D., Poynard, T., Tursz, T., Raposo, G., 
Yagita,  H.,  Ryffel,  B.,  Kroemer,  G.,  Zitvogel,  L.,  ‘A 
novel dendritic cell subset involved in tumor im-
munosurveillance’, Nat Med, 2006, Feb;12(2):214-9. 

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Coordinator 

JonathanMAustyn
University of Oxford
John Radcliffe Hospital
Nuffield Department of Surgery 
Headington
Oxford OX3 9DU, UK 
E-mail (Project Manager): miriam.mendes@nds.ox.ac.uk  

Partners 

CarlG.Figdor
University Hospital
Medical Centre Nijmegen
Nijmegen, Netherlands

MurielMoser
Université Libre de Bruxelles
Brussels, Belgium

AnneO’Garra
Medical Research Council
London, UK

FrancescaGranucci
University of Milano-Bicocca
Milan, Italy

GeroldSchulerandAlexanderSteinkasserer
Friedrich Alexander Universitat
Erlangen, Germany

AndreaSplendiani
Leaf Bioscience S.R.L.
Milan, Italy

CharlesNicolette
Argos Biosciences, Inc
Durham, US

FilippoPetralia
Sekmed S.R.L.
Milan, Italy

SebastianAmigorena
Institut Curie
Paris, France

ThierryBoonandPierreCoulie
Christian de Duve Instite of Cellular Pathology
Brussels, Belgium

DuccioCavalieri
Preclinica e Clinica Università degli studi di Firenze
Dipartimento Di Farmacologia
Florence, Italy

SandraGessani
Istituto Superiore di Sanita
Rome, Italy

NicolasGlaichenhaus
Universite Nice-Sophia Antipolis
INSERM
Valbonne, France

RobertCoffin
Biovex
Abingdon, UK

CatherineDeGreef
BruCells SA
Brussels, Belgium

UgoD’Oro
Chiron Vaccines S.R.L.
Siena, Italy

RolfKiessling,HakanMellstedt,PavelPisa
Karolinska Institutet
Stockholm, Sweden

CornelisJ.M.Melief
Leiden University Medical Centre
Leiden, Netherlands

PhilippePierre
Centre CNRS-INSERM d’Immunologie de Marseille-Luminy
Marseille, France

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dC-THERA

MariaPiaProtti
Fondazione Centro San Raffaele Del Monte Tabor
Milan, Italy

MatthiasMann
Max-Planck Institute of Biochemistry
Martinsried, Germany

AndreasRadbruch,AlexanderScheffold,
AndreasThiel
Deutsche Rheuma-Forschungszentrum Berlin
Berlin, Germany

AlbertoMantovani
Istituto Clinico Humanitas
Milan, Italy

CaetanoReiseSousa
Cancer Research UK
London, UK

BeneditaRocha
Université René Descartes
Faculté de Médecine Necker
Paris, France

AntonioLanzavechia,MarkusManz,
FedericaSallusto,MariagraziaUguccioni
Insitute for Research in Biomedicine
Bellinzona, Switzerland

MarkSuter
University of Zurich
Zurich, Switzerland

KrisThielemans
Vrije Universiteit Brussel
Brussels, Belgium

HermannWagner
Technische Universitaet Muenchen
Munich, Germany

LaurenceZitvogel
Institut Gustave Roussy
Paris, France

PaolaRicciardi-Castagnoli
Genopolis – Consortium for Functional Genomics
Milan, Italy

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dC-vACC

Therapeuticin vivoDNArepairbysite-specificdouble-strandbreaks

ContractNo 
Projecttype

LSHB-CT-2003-03037
SpecificTargetedResearchProject

ECcontribution  e 2000000

Startingdate 1January2004
Duration 36months






Background and objectives:

immune  system  of  vertebrate  animals 
The 
evolved  to  respond  to  different  types  of  per-
turbations,  such  as  pathogens,  whilst  limiting 
self-tissue damage. Initiation of the immune re-
sponse is accomplished by unique antigen pre-
senting  cells  called  dendritic  cells  (DC)  –highly 
phagocytic sentinels of the immune system, rest-
ing until they encounter foreign microorganisms 
or inflammatory stimuli. 

Early-activated  DC  trigger  innate  immune  re-
sponses  that  represent  the  first  line  of  defence 
against  pathogens  and  provide  effective  anti-
tumour  immunity.  Activated  DC  subsequently 
prime  antigen-specific  immune  responses  (T 
and  B  lymphocytes)  to  clear  the  infections  and 
give  rise  to  immunological  memory.  Activation 
of  Natural  Killer  (NK)  cells  is  also  key  during  an 
anti-pathogen response. A direct involvement of 
NK cells has also been shown in anti-tumour re-
sponses  in  different  systems  and  unequivocally 
observed in patients with cancer. 

The aim of DC-VACC, a Specific Targeted Research 
project  (STREP),  was  to  develop  novel  vaccine 
technologies and to use DC as natural adjuvants 
with specificity and minimum side effects. Early 
clinical trials have shown that antigen-pulsed DC 
have potential in the treatment of cancer – also 
applicable  in  the  eradication  of  infectious  dis-
eases. The objective of the proposed project was 
part  of  the  priority  area  1.2.4-6:  ‘Development 

of  Vaccine  Technologies  Targeted  to  Dendritic 
Cells’,  proposed  as  a  STREP  for  the  Framework 
Programme 6. The basis of this proposal was to 
develop in situ DC targeting for use as vaccines 
in infectious diseases and cancer.

Approach and methodology:

The  consortium  has  effectively  generated  im-
proved  reagents  and  protocols  for  antigen  de-
livery  and  targeting,  which  improved  antigen 
processing  and  presentation  by  DC  and  can  be 
used  for  vaccine  and  therapeutic  technology. 
They  have  also  defined  optimal  reagents  and 
protocols for maturation and activation of mouse 
and human DC in vitro for use in vaccination and/
or therapeutic intervention. Optimisation of pro-
tocols was compared across both species, which 
is essential for use in preclinical models and clini-
cal trials in future NoEs and/or IPs.

Main findings:

DC-VACC  targeted  two  specific  objectives  in 
Work Packages (WP) 1 and 2.

WP1 generated the tools and methods required 
for appropriate and efficient targeting and anti-
gen delivery for the development of DC vaccine 
technology.  This  included  the  construction  of 
viral  and  bacterial  vectors,  modification  of  RNA, 
peptides  and  proteins,  and  antibody  develop-
ment  for  specific  targeting  of  DC  receptor  rep-
ertoire. Excellent research progress was made in 

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the following:

•  microbial  vectors  development  (bac-
terial and viral);
•  modification  of  RNA,  peptides  and 
proteins;
•  antibody development for specific tar-
geting of DC receptor repertoire

WP2 defined optimal reagents and protocols for 
maturation and activation of mouse and human 
DC  in  vitro  for  use  in  vaccination  and/or  thera-
peutic  intervention,  such  that  optimisation  of 
protocols for both species are compared so as to 
promptly  facilitate  information  from  preclinical 
models being transferred rapidly to clinical trials 
in  future  NoEs  and/or  IPs. The  DC-VACC  consor-
tium determined the following:

signals  for  activation/maturation  of 

• 
mouse and human DC; 
•  optimised  human  DC-vaccines:  rea-
gents and procedures;
• 
human DC;
•  DC-based bioassays.

transcriptome  analysis  of  mouse  and 

Time (min)

The humanised antibody to DC-SIGN hD1 G2/G4 (hD1) 
was cross-linked to a model antigen, keyhole limpet 
hemocyanin (KLH). Using confocal microscopy it was 
observed that the chimeric antibody-protein complex 
(hD1-KLH).bound specifically to DC-SIGN, was rapidly 
internalised and translocated to the lysosomal compart-
ment as shown in the above figure over time (minutes).

There  has  been  great  productivity  with  respect 
to the development of microbial vectors, and the 
ability to deliver antigen and target DC, as well as 
approaches. These include the discovery of new 
pathways to target the enhancement of anti-tu-
mour  therapy  and  therapeutic  intervention,  as 
well as for vaccination in infectious diseases. 

The knowledge from this project has been widely 
disseminated via publications and international 
immunology  conferences.  During  the  duration 
of  the  project,  meetings  (held  together  with  an 
NoE, DC-THERA) were held at various sites to ex-
change scientific knowledge and via the website 
created by Partner Biopolo (http://www.biopolo.
it/), where the list of publications generated from 
DC-VACC are displayed.

Ultimately, the studies from DC-VACC have result-
ed in the discovery of new adjuvant approaches. 

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IMMUNOTHERAPY ANd TRANSPLANTATION

Boonstra, A., Rajsbaum, R., Holman, M., Marques, 
R., Asselin-Paturel, C., Pereira, J.P., Bates, E.M., Akira, 
S., Vieira, P., Liu, Y-J., Trinchieri, G., O’Garra, A. ‘Mac-
rophages and myeloid DC, but not plasmacytoid 
DC,  produce  IL-10  in  response  to  MyD88-  and 
TRIF- dependent TLR signals, and TLR-independ-
ent signals’, J.Immunol, 2006, 177, 7551 - 7558.

Major publications

Smith, C.L., Dunbar, P.R., Mirza, F., Palmowski, M.J., 
Shepherd, D., Gilbert, S.C., Coulie, P., Schneider, J., 
Hoffman, E., Hawkins, R., Harris, A.L., Cerundolo, V., 
‘Recombinant  modified  vaccinia  Ankara  primes 
functionally activated CTL specific for a melano-
ma tumor antigen epitope in melanoma patients 
with a high risk of disease recurrence’, Int J Cancer, 
2005, Jan 10;113(2):25.

Hermans, I., Silk, J., Gileadi, U., Masri, H.S., Shepherd, 
D., Farrand, K.J., Salio, M., Cerundolo, V., ‘Dendritic 
cell  function  can  be  modulated  through  co-op-
erative actions of TLR ligands and invariant NKT 
cells’, J. of Immunology, 2007, Mar 1; 178(5):2721-9.

Granucci, F., Zanoni, I., Pavelka, N., van Dommelen, 
S.L.H., Andoniou, C.E., Belardelli, F., Degli Esposti, 
M.A.,  Ricciardi-Castagnoli,  P.,  ‘A  Contribution  of 
Mouse Dendritic Cell-Derived IL-2 for NK Cell Ac-
tivation’, J. Exp. Med., Aug 2004; 200: 287-295.

Foti, M., Granucci, F., Pelizzola, M., Beretta, O., Ric-
ciardi-Castagnoli,  P., ‘Dendritic  cells  in  pathogen 
recognition and induction of immune responses: 
a  functional  genomics  approach’,  J  Leukoc  Biol, 
2006, May;79(5):913-6. Review.

Schaft, N., Dorrie, J., Thumann, P., Beck, V.E., Mull-
er,  L.,  Schultz,  E.S.,  Kampgen,  E.,  Dieckmann,  D., 
Schuler,  G., ‘Generation  of  an  optimized  polyva-
lent monocyte-derived dendritic cell vaccine by 
transfecting  defined  RNAs  after  rather  than  be-
fore  maturation’,  J.  Immunol,  2005,  174  (5):3087-
3097.

Prechtel,  A.T., Turza,  N.M.,  Kobelt,  D.J.,  Eisemann, 
J.I., Coffin, R.S., McGrath, Y., Hacker, C., Ju, X., Zenke, 
M., Steinkasserer, A., ‘Infection of mature dendrit-
ic cells with herpes simplex virus type 1 dramati-
cally  reduces  lymphoid  chemokine-mediated 
migration’, J Gen Virol, 2005, Jun;86(Pt 6):1645-57.

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dC-vACC

Coordinator

AnneO’Garra
National Institute for Medical Research Council
The Ridgeway
Mill Hill
London NW7 1AA, UK
E-mail: aogarra@nimr.mrc.ac.uk

Partners

LeonardoBiondi
Biopolo SCRL
Milan, Italy

VicenzoCerundolo
University of Oxford
Oxford, UK

CarlFigdor
University Medical Centre
Nijmegen, Netherlands

MurielMoser
Université Libre de Bruxelles
Brussels, Belgium

PaolaRicciardiCastagnoli
University of Milano-Bicocca
Milan, Italy

GeroldSchuler
University Hospital of Erlangen
Erlangen, Germany

FilippoPetralia
Sekmed Srl
Milan, Italy

KaiZwingenberger
Brucells SA
Brussels, Belgium

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THERAvAC

Optimiseddeliverysystemforvaccinestargetedtodendriticcells

ContractNo 
Projecttype

LSHB-CT-2004-0382
SpecificTargetedResearchProject

ECcontribution  e 2267000

Startingdate 1March2004
Duration 4months






Background and objectives:

In response to urgent medical and societal needs 
for novel immunotherapies for cancer and chronic 
infections, as well as for prophylactic vaccination, 
optimised  delivery  systems  for  vaccines  target-
ing dendritic cells will be developed and clinically 
evaluated. The approach relies on two new anti-
gen delivery vectors: the detoxified adenylate cy-
clase toxoid (ACT) and the porcine parvovirus-like 
particles (PPV-VLP), which were recently shown to 
target dendritic cells very efficiently and specifi-
cally, allowing for a highly effective presentation 
of delivered antigens to T cells. 

These  vaccine  vectors  enable  the  induction  of 
strong, specific and protective immune respons-
es, and have an established record of safety and 
efficacy in preclinical animal models. Under this 
project,  academic  experts  in  immunology,  vac-
cinology,  and  molecular  and  structural  biology 
have  joined  forces  with  toxicologists,  clinicians 
and vaccine production experts from two com-
panies, to translate these novel vaccine technolo-
gies from research into clinical application. 

Based  on  the  preclinical  record  of  ACT-based 
vaccines  in  animal  models,  the  step  into  the 
safety and efficacy phase I/II human trial will be 
made  with  an  ACT-based  construct  delivering 
the tyrosinase A.2 epitope as a therapeutic vac-
cine  for  metastasic  melanoma.  Simultaneously 
with Good Manufacturing Practice (GMP) batch 
production, development and clinical testing, an 

Electron microscopy image of PPV-VLP particles, one of 
the two vaccine delivery vehicles developed by THERAVAC

in-depth  analysis  of  the  cellular  and  molecular 
mechanisms, as well as of the structural basis of 
ACT interaction with dendritic cells will be con-
ducted.  Particular  emphasis  will  be  placed  on 
gaining  new  knowledge  for  furthering  the  de-
livery capacity of the ACT molecule towards en-
hanced efficiency and broader versatility in clini-
cal use. The PPV-VLP vector will be developed in 
parallel, by defining its cellular receptor and traf-
ficking  inside  dendritic  cells,  preclinical  efficacy 
and toxicology, in order to bring this alternative 
vaccine carrier to the level of clinical trial maturity. 

Approach and methodology:

THERAVAC  combines  basic  research  into  the 
mechanism  of  function  of  these  novel  vaccine 
targeting systems with their preclinical applica-
tion, including a phase I/II trial. The first two work 
packages form a demonstration activity centred 
on the production of the GMP lot and the han-
dling  of  the  phase  I/II  trial.  The  remaining  four 

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THERAvAC

work  packages  correspond  to  the  research  and 
development  component  of  the  project,  aimed 
at  understanding  the  basic  mechanisms  at  the 
molecular level. The latter work package employs 
cell biology, immunology, biochemistry and bio-
physics,  including  structural  biology,  to  address 
the  questions  of  specificity  and  efficacy  of  the 
vaccine vehicle molecules used.

Expected outcome:

THERAVAC aims to achieve:

the detailed toxicology assessment of 

•  production of a GMP batch of ACT car-
rying  the  melanoma  tyrosinase  epitope 
suitable for a phase I/II trial; 
• 
this GMP batch of ACT;
•  a phase I/II trial in melanoma patients 
of this GMP batch of ACT carrying the ty-
rosinase melanoma CD8+ T cell epitope; 
• 
the  understanding  of  the  interaction 
of  ACT  with  dendritic  cells  at  molecular 
and atomic details;
• 
the  detailed  understanding  of  the 
mechanism  of  PPV-VLP  specificity  and 
efficacy;
improved  vac-
• 
cine delivery molecules based on our im-
proved understanding of their functional 
mechanism.

the  engineering  of 

IMMUNOTHERAPY ANd TRANSPLANTATION

These various activities associated with the ACT 
molecule  synergise  to  bring  about  the  potency 
of  ACT  as  an  antigen  delivery  vector  into  den-
dritic cells and induction of specific T cell medi-
ated  responses  against  delivered  antigens.  The 
complex  activity  of  ACT  is  dissected  into  indi-
vidual contributions by characterising properties 
of ACT variants, which are selectively blocked by 
specific  mutations  in  individual  steps  of  toxoid 
penetration  into  cells  and  in  channel-forming 
and signalling properties of ACT. A novel mecha-
nism  of  calcium  mobilisation  into  myeloid  cells 
by ACT was discovered, and shown to be linked 
to  AC  domain  translocation  across  cytoplasmic 
membrane  of  cells.  Over  the  past  36  months, 
important progress was made in understanding 
the  mechanisms  underlying  the  toxoid  interac-
tion  with  CD11b,  signalling  activity  on  myeloid 
cells and the capacity to deliver the AC domain 
with antigens into cells. This was efficiently used 
to  manipulate  the  antigen  delivery  potency  of 
toxoid forms of ACT.

Important progress was also made in the under-
standing  of  the  mechanisms  of  how  PPV-VLPs 
interact  with  dendritic  cells.  Importantly,  these 
VLPs  were  shown  to  possess  a  strong  adjuvant 
activity. THERAVAC demonstrates that the bacu-
lovirus (BV) is responsible for this adjuvant effect 
and that it plays a major role in the strong immu-

Main findings:

The  Bordetella  adenylate  cyclase  toxin  (ACT) 
binds the complement receptor 3 (CD11b/CD18, 
CR3 or Mac-1) of host myeloid phagocytes, pen-
etrates  their  cytoplasmic  membrane,  and  upon 
translocation  into  cytosol  and  binding  of  cal-
modulin,  it  delivers  into  the  MHC  Class  I/II  anti-
gen  presentation  pathway  its  AC  domain.  Fur-
thermore,  ACT  can  form  small  cation  selective 
channels  in  target  membranes,  bind  calcium, 
promote its influx into cells and induce a cascade 
of signalling events leading to the maturation of 
dendritic cells. 

Scheme of antigen delivery by recombinant ACT. 
The toxoid inserts into cellular membrane of antigen 
presenting cells in two different conformations that give 
rise to molecules with the AC domain translocated into cell 
cytosol and to molecules forming oligomeric membrane 
channels with the AC domain stuck at the external face of 
cellular membrane. The translocated AC domain bearing the 
inserted antigen is processed inside the cytosol of antigen 
presenting cells by proteasome. The peptides are then 
transported by TAP into the endoplasmic reticulum and bind 
to newly synthesised MHC I molecules. The MHC I – peptide 
complexes are transported to the cell surface and pre-
sented to CD8+ T cells, thereby promoting their activation. 
Endocytosis of membrane associated CyaA may also occur 
and CyaA molecules can be processed to antigenic peptides 
that bind to MHC II molecules. These MHC II complexes are 
presented to CD4+ T cells. Reviewed in Simsova et al. (2004) 
Int. J. Med. Microbiol. 293, 571-576. © Urban & Fischer Verlag.

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IMMUNOTHERAPY ANd TRANSPLANTATION

nogenicity of PPV-VLPs produced in the baculo-
virus-insect cell expression system. This adjuvant 
behaviour of BVs is mediated primarily by IFNΔ/Δ, 
although mechanisms independent of type I in-
terferon signalling are also involved.

The first pre-GMP batch has been prepared on a 
small scale by CellGenix, GmbH, the subcontrac-
tor  chosen  for  this  task.  Once  this  batch  is  vali-
dated, a GMP-like batch will be provided for toxi-
cology, as well as a larger-scale GMP preparation 
for the clinical trial, starting in 2008.

Basler, M., Masin, J., Osicka, R., Sebo, P., ‘Pore-form-
ing  and  enzymatic  activities  of  Bordetella  ade-
nylate  cyclase  synergise  in  promoting  lysis  of 
monocytes’, Infect Immun, 2006, 74, 2207-14.

Mascarell,  L.,  Bauche,  C.,  Fayolle,  C.,  Diop,  O.M., 
Dupuy,  M.,  Nougarede,  N.,  Perraut  R.,  Ladant,  D., 
Leclerc,  C., ‘Delivery  of  the  HIV-1  Tat  protein  to 
dendritic  cells  by  the  CyaA  vector  induces  spe-
cific Th1 responses and high affinity neutralising 
antibodies in non human primates’, Vaccine, 2006, 
24, 3490-9.

Major publications
a) Publications in peer-reviewed journals

Prior, S., Fleck, R. A., Gillett, M., Rigsby, P., Corbel, M. 
J., Stacey, G., Xing, D. K. L., ‘Evaluation of adenylate 

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IMMUNOTHERAPY ANd TRANSPLANTATION

THERAvAC

cyclase toxin constructs from Bordetella pertus-
sis  as  vaccine  candidate  antigens  in  an  in  vitro 
complement-dependent opsonophagocytic-kill-
ing model’, Vaccine, 2006, 24:4794-4803.

Cheung, Y.C.G., Xing, D., Prior, S., Corbel, M.J., Par-
ton,  R.,  Coote,  J.G.,  ‘Adjuvant  effect  of  different 
forms  of  adenylate  cyclase  toxin  of  Bordetella 
pertussis on protection afforded by acellular per-
tussis  vaccine  in  a  murine  model.  Infect.  Immu, 
2006, 74:6797-6805.

Bauche, C., Chenal, A., Knapp, O., Bodenreider, C., 
Benz,  R.,  Chaffotte,  A.,  Ladant,  D., ‘Structural  and 
functional  characterisation  of  an  essential  RTX 
sub-domain of Bordetella pertussis adenylate cy-
clase toxin’, J Biol Chem, 2006, 281:16914-16926.

Fiser, R., Masin, J., Basler, M., Krusek, J., Spulakova, 
V.,  Konopasek,  I.,  Sebo,  P., ‘Third  Activity  of  Bor-
detella Adenylate Cyclase (AC) Toxin-Hemolysin: 
Membrane translocation of AC domain polypep-
tide promotes calcium influx into CD11b+ mono-
cytes independently of the catalytic and hemo-
lytic activities’,  J. Biol. Chem, 2007, 282 2808-2820.

Vojtova, J., Kamanova, J., Sebo, P., Bordetella ade-
nylate cyclase toxin: a swift saboteur of host de-
fense. Curr. Op. Microbiol, 2006, 9, 69-75.

Hervas-Stubbs, S., Rueda, P., Lopez, L., Leclerc, C., 
Insect  baculovirus  strongly  potentiate  adap-
tive 
induc-
immune  responses  through  the 
tion  of  type  1  interferon.  J.  Immunol,  2007,  Feb 
15;178(4):2361-9.

b) Patents

Leclerc,  C.,  Boisgerault,  F.,  Rueda,  P., ‘Empleo  de 
partículas  pseudovirales  como  adyuvante.  No. 
P200403077’ Presented in the Spanish Patent and 
Trademark Office, 23/12/2004. 

Leclerc, C., Hervas-Stubbs, S., Rueda, P., Lopez, L.. 
Viral adjuvants. EP 06380240.9, 1.09.06 

Coordinator

ClaudeLeclerc
Institut Pasteur
25 rue du Dr. Roux 
75014 Paris, France
E-mail: cleclerc@pasteur.fr

Partners

AnitaLewit-Bentley
CNRS
Cachan, France

RinoRappuoli
Novartis
Sienna, Italy

PalomaRueda
INGENASA
Madrid, Spain

PeterSebo
Institute of Microbiology
Prague, Czech Republic

DanielLadant
Institut Pasteur
Paris, France

BenoîtVandenEynde
ICP
Brussels, Belgium

DorothyXing
NIBSC
Potters Bar, UK

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IMMUNOTHERAPY ANd TRANSPLANTATION
dENdRITOPHAgES

Therapeuticcancervaccines

ContractNo 
Projecttype

LSHB-CT-2003-0383
SpecificTargetedResearchProject

ECcontribution  e 1999940

Startingdate 1January2004
Duration 42months






Background and objectives:

Cancer  has  been  considered  as  a  fatal  disease 
for  many  years  and  has  been  selected  as  a  pri-
ority  disease  throughout  successive  European 
research programmes. Cancer is a group of over 
100  related  diseases  characterised  by  uncon-
trolled cell growth that expands locally by inva-
sion and systemically by metastases. Cancer is the 
second most common cause of death in western 
countries accounting for 2.5 million deaths in the 
developed world. 

Existing  therapies  have  been  able  to  increase 
the  survival  rate,  though  only  marginally.  Cur-
rent  cancer  treatments  allow  for  the  treatment 
of approximately 50% of the patients, but these 
therapies are associated with potentially serious 
or intolerable adverse side effects. Thus, the qual-
ity of life of cancer patients is presently not sat-
isfactorily preserved with surgery, chemotherapy 
and  radiotherapy.  However,  the  advent  of  new 
therapies,  such  as  cancer  vaccines,  is  expected 
to enhance the quality of life since the dendritic 
cell  (DC)  vaccines  present  an  alternative  immu-
notherapy  approach  with  very  few  side  effects. 
Quality of life is therefore preserved during treat-
ment and no hospitalisation is required.

transformed into effector monocyte-derived DCs 
(dendritophages)  which  fight  the  patient’s  own 
disease. The therapeutic cell drug comprises DCs 
loaded with cancer-specific antigens to activate 
the  patient’s  immune  system  after  re-injection. 
This ex vivo approach is compared to the in vivo 
injection of the tumour antigen plus adjuvant.

This  project  aims 
to  demonstrate  on  a 
multinational  level,  the  immunogenicity  and 
efficacy,  as  well  as  the  reproducibility  and 
feasibility  of  anticancer  vaccine  by  sequential 
steps: choosing the best DC vaccination strategy 
through  adequate  pre-clinical  studies 
(DC 
differentiation and maturation, tumour antigens 
selection  and  loading,  dose  delivered,  and  site 
and  vaccination  schedule);  and  monitoring  the 
immune  response  in  correlation  to  the  clinical 
response  after  defining  the  most  relevant 
immuno-monitoring techniques. 

This requires the establishment of quality control 
criteria, the design for the production of the cel-
lular  product,  as  well  as  for  the  proteic  tumour 
antigen  and  its  formulation,  and  to  optimise  a 
GMP process. DENDRITOPHAGES initiated a clini-
cal trial to evaluate the vaccines on progressing 
prostate cancer patients.

The  goal  of  cancer  therapeutic  cell  vaccine  is 
to  prevent  progression  and  tumour  recurrence. 
Adoptive therapy in adjuvant settings will com-
plement  classical  anti-cancer  treatments.  In  this 
technology  the  patient’s  blood  monocytes  are 

On a short-term basis, the new treatments direct-
ed  to  residual  cancer  diseases  resistant  to  con-
ventional treatments will be used in an adjuvant 
setting  as  second  line  therapy.  On  a  long-term 
basis,  these  patient-oriented  therapeutic  strate-

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IMMUNOTHERAPY ANd TRANSPLANTATION

Investigations in mouse models and recent evi-
dence  from  the  use  of  the  cell  therapeutic  vac-
cine to treat and  prevent cancer recurrence will 
therefore  be  comprised  of  adequately  differ-
entiated  and  maturated  DCs  derived  from  the 
patient’s  blood  monocytes  loaded  in  vitro  with 
tumour  antigens.  The  resulting  cellular  vaccine 
is re-injected into the patient to stimulate an im-
mune response against their cancer. In a clinical 
setting, DC should be differentiated ex vivo, load-
ed  with  the  chosen  antigenic  formulation,  and 
finally, maturation should be initiated by a short 
treatment. 

DENDRITOPHAGES anticipates that, once injected, 
DC will migrate to T cell areas during the sponta-
neous  progression  of  their  activation  and  repro-
duce the physiological process of maturation.

The goal of these therapeutic vaccines is to pre-
vent  metastasis  development  and  to  provide 
long-term  protection.  This  immunotherapeutic 
approach can be applied to diseases other than 
cancer, in particular to stimulate an immune re-
action against one or several antigens associated 
with virus-infected cells. 

In  view  of  the  urgent  need  to  provide  adapted 
therapy  to  treat  cancer,  improved  standardised 
DC  protocols  will  enable  rationally  designed 
clinical  studies  to  give  cancer  vaccine  immuno-
therapy further credibility and cancer patients a 
new therapeutic option associated with a better 
quality of life. Sequential steps and objectives are 
required.

Approach and methodology:

The methodology developed and optimised dur-
ing the project is adoptive cancer vaccine where 
DCs  are  differentiated  from  apheresis  of  mono-
nuclear cells, then matured and loaded with tu-
mour antigen before being purified, frozen, aliq-
uoted and re-injected into the patients.
The participating teams compared a number of 

Dendritophage Copyright: IDM Author: J. Davous

gies should replace some first line conventional 
treatments of cancer.

Dendriticcells

Discovered  in  the  early  1970s  by  Steinman  and 
Collin,  DCs,  key  actors  of  the  immune  system, 
are  now  considered  as  being  the  most  potent 
adjuvant  for  cancer  immunotherapy  to  present 
and  stimulate  specific T  cell  responses.  Capable 
of taking up the antigen at the pathogen entry 
site, to migrate into secondary lymphoid tissues 
and to activate both helper and cytotoxic T cells, 
DC  can  also  interact  with  B  cells,  and  probably 
natural killer cells, and thus direct the specificity 
of the immune response. 

However, it is only upon maturation that they ac-
quire full expression of costimulatory molecules 
and  major  histocompatibility  peptide  complex-
es;  increase  cytokine  production;  and  migrate 
to  draining  lymphoid  organs.  Despite  a  grow-
ing interest in the use of autologous DC  for the 
management  of  cancer  and  infectious  diseases, 
the relation between the type of DC  infused in 
humans  and  the  type  of  immune  response  ob-
tained, is not yet clear. 

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IMMUNOTHERAPY ANd TRANSPLANTATION

differentiation and maturation methods, and se-
lected  IL-13,  GM-CSF  and  then  IFNg  +  FMKp  as 
GMP  processes,  thus  producing  more  than  one 
billion DCs. 

KSA (Epcam) was chosen as a tumour proteic an-
tigen  and  loaded  on  DCs  as  a  protein,  encaplu-
lated in liposomes or in virosomes. The stimula-
tion of KSA specific cytotoxic T lymphocytes by 
these DCs  was achieved. Such loaded DCs have 
already been injected in prostate cancer patients 
in Vienna, and will be compared to the KSA-lipo-
somes subcutaneous vaccine.

Main findings:

DENDRITOPHAGES  has  already  finalised  many 
steps  required  for  the  evaluation  of  an  antican-
cer vaccine:

standardised  preparation,  differentia-
• 
tion and maturation of DCs: three distinct 
processes  for  DC  differentiation  (IL-13  + 
GM-CSF 7 days; IL-4 + GM; IFN Δ 3 days) and 
maturation factors were compared by five 
partners  for  differentiated  DCs,  matured 
or not. DCs differentiated in the presence 
of IL-13 appear to be the most robust in a 
GMP process and the only ones to secrete 
high  IL-12.  Maturation  is  induced  within 
six hours of being in the presence of IFNg 
and  of  Klepsiella  bacterial  membrane 
fraction.
•  KSA is the choice of the adequate anti-
gen: The KSA (EpCam) antigen was chosen 
as  a  pancarcinoma  tumour  Ag  protected 
by IDM I.P.
•  antigen loading: Loading of immature 
DCs with KSA liposomes/KSA, virosomes/
KSA,  and  following  a  24-hour  stimula-
tion  period  of T8  expansion,  the  findings 
showed  that  virosomes  were  effective 
only  in  stimulating  CD4 T  cells.  KSA  lipo-
somes were the most effective in inducing 
a CD8 T cell response. They were therefore 
selected for clinical approach.

injection site: The migration of imma-
• 
ture versus mature DCs were followed in 
patients according to the injection route, 
using  Cu64  labelled  DCs.  Intradermal  in-
jection  delivers  a  better  accumulation  in 
lymph nodes. The tracking and migration 
of  DCs  were  also  studied  in  mice  for  the 
different  DC  types  produced.  They  were 
labelled with Ittrium 86.
the development of an emulsion with 
• 
adjuvant:  Loading  with  liposomes  also 
containing lipid A as adjuvant and resus-
pended in water in oil emulsion appeared 
the  most  effective,  in  comparison  with 
KSA alone or in virosomes.
• 
the 
immunological  monitoring  of 
response  to  the  vaccine:  Specific  CD8  T 
cell  and  antibody  responses  to  KSA  are 
measured.
•  generation  of  a  GMP  process  accept-
able  by  regulatory  authorities:  Based  on 
the  GMP  industrial  cellular  process  de-
veloped  by  IDM,  DENDRITOPHAGES  de-
termined the optimal strategy of DCs im-
munotherapy against prostate cancer. The 
DC vaccine  was elaborated and tested in 
phase I/II studies on prostate cancer using 
the selected autologous DCs loaded with 
the  KSA  antigen.  The  researchers  evalu-
ated  its  tolerance  and  immune  response 
generated  against  KSA,  and  looked  for 
signs  of  clinical  vaccine  efficiency,  prob-
ably based on serum PSA level and evolu-
tion.  A  second  study  with  liposomal  KSA 
plus  adjuvant  was  initiated  in  prostate 
cancer patients for comparison.

GMP  processes  and  clean  room  for  cell  therapy 
were achieved in Paris, Rome, Melbourne and Vi-
enna. A GMP lot of KSA incorporated in liposomes 
was prepared, qualified and released for clinical 
use. A phase I clinical trial initiated in Vienna by 
the end of 2005 on advanced prostate cancer pa-
tients compared the immune response obtained 
after vaccination with DCs loaded with free KSA 

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dENdRITOPHAgES

IMMUNOTHERAPY ANd TRANSPLANTATION

or with a liposomal KSA-lipid A emulsion. A sec-
ond  clinical  trial  was  initiated  in  Regensburg 
on  less-advanced  prostate  cancer  patients  vac-
cinated  with  liposomal  KSA  emulsion  plus  lipid 
A. The duration of the project was extended by 
six  months  to  launch  this  clinical  protocol  after 
regulatory approval.

Coordinator

JacquesBartholeyns
IDM SA Immuno-Designed Molecules
172 rue de Charonne
75011 Paris, France
E-mail: jb@idm-biotech.com

Partners

MilesPrince
Centre for Blood Cell Therapies
c/o Peter MacCallum Cancer Centre
Victoria, Australia

RheinardGlueck
Etnabiotech
c/o Università di Catania
Facoltà di Medicina
Dipartimento di Farmacologia Sperimentale e Clinica
Catania, Italy

ThomasFelzmann
Children’s Cancer Research Institute
Vienna, Austria

FilippoBelardelli
Istituto Superiore di Sanità
Laboratori di Virologia
Rome, Italy

AndreasMackensen
University of Regensburg
Department of Haematology/Oncology
Regensburg, Germany

Anne-CéciledeGiacomoni
Alma Consulting Group
Lyon, France

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IMMUNOTHERAPY ANd TRANSPLANTATION
gENOMES TO vACCINES

Translatinggenomeandproteomeinformationintoimmunerecognition

ContractNo 
Projecttype

LSHB-CT-2003-03231
SpecificTargetedResearchProject

ECcontribution  e 2000000

Startingdate 1January2004
Duration 48months

Website  www.cbs.dtu.dk/researchgroups/



immunology/Genomes2vaccines/index.php








Background and objectives:

Genome information is accumulating rapidly and 
it encompasses a wide range of species, includ-
ing humans and many pathogens, among others. 
It is now possible to completely sequence micro-
bial genomes within a period ranging from days 
to months, and genomics can therefore be used 
as a powerful starting point to investigate the bi-
ology of any pathogen of interest. The translated 
protein sequence information constitutes a sig-
nificant input to the immune system. 

Indeed,  the  immune  system  considers  peptides 
as one of its key targets, and it has devoted an en-
tire arm — that of T cells, which essentially control 
specific  immune  responsiveness  —  to  peptide 
recognition. T cells are specific for peptides pre-
sented in the context of major histocompatibil-
ity complexes (MHC). Prior to their presentation, 
these peptides were generated in the cytosol by 
limited proteolytic fragmentation of all available 
protein antigens, then translocated to the endo-
plasmic  reticulum  (ER)  and  specifically  sampled 
by the MHC for subsequent presentation. 

The MHC is extremely polymorphic and the pep-
tide  binding  specificity  varies  for  the  different 
polymorphic  MHC  molecules.  The  net  effect  of 
this  complicated  system  is  that  each  individual 
presents to their T cells a unique and highly di-
verse  peptide  imprint  of  the  ongoing  protein 
metabolism. The scientific rationale of Genomes 
To Vaccines is one of understanding, describing 

and predicting how the immune system handles 
proteins;  or  more  specifically,  of  how  it  gener-
ates, selects and recognises peptides. Generating 
these predictive tools amounts to translating ge-
nomes/proteomes into immunogens, and it will 
enable a new and rational approach for vaccina-
tion and immunotherapy. 

Approach and methodology:

Genomes  To  Vaccines  focuses  on  MHC  class  I 
mediated  antigen  presentation  to  cytolytic  T 
cells. While  several  mechanisms  involved  in  an-
tigen  processing  and  presentation  have  been 
described  in  general  terms,  only  a  few  of  them 
have been described in sufficient detail to allow 
for the accurate prediction of their outcome; it is 
a key requirement so as to achieve an overall (in-
tegrated) prediction of the final result of a series 
of processing events. 

To  produce  ample  and  accurate  data,  Genomes 
To  Vaccines  will  generate  biochemical  high-
throughput  assays,  representing  the  most  im-
portant  events  in  antigen  processing  and  pres-
entation.  Focused  on  peptides  and  peptide 
recognition, the research team seeks to develop 
a  high  throughput  peptide-array  technology, 
and  it  aims  to  generate  accurate  and  quantita-
tive predictions through the following data-driv-
en approach: 

•  build  and  extend  databases  contain-
ing  information  on  natural  ligands  and 
immune epitopes; 

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•  use  these  databases  to  generate  pre-
liminary predictions; 
•  use these predictions to select a set of 
data points that will complement existing 
data  (i.e.  the  most  information-rich  new 
data); 
•  perform  the  experiment  to  actually 
generate the new quantitative data; 
•  add  the  new  data  to  the  existing 
databases; 
•  update and improve the bioinformat-
ics resources.

ing  one  of  the  bottlenecks  in  the  current  state-
of-the-art  search  for  vaccine  candidates  and 
targets for immunotherapy. Only with computa-
tional  bioinformatics  tools  and  concurrent  high 
throughput  life-science  tools  will  it  be  possible 
to search entire genomes for the presence of im-
munogenic epitopes and validate them. Eventu-
ally, it will enable a rational approach to popula-
tion-wide  vaccination,  as  well  as  individualised 
immunotherapy. 

Main findings:

The  approach  constitutes  an  iterative  and  in-
teractive  loop  between  bioinformatics  and  wet 
biochemistry,  leading  to  successively  improved 
predictions. Eventually, this will allow Genomes_
To_Vaccines to search the entire sequence space 
(more than 1012 members) computationally (fast 
and  cheap),  while  reserving  the  experimental 
work (slow and costly) to the most information-
rich data points. The researchers believe that this 
integrated  and  iterative  approach  will  generate 
accurate prediction algorithms with a minimum 
of effort (i.e. in the most cost-efficient manner). 

Expected outcome:

Genomes To Vaccines has generated efficient bi-
ochemical assays for three different major steps 
in the generation of T cell epitopes. This includes 
the proteolytic fragmentation of protein antigens 
(proteasome),  the  peptide  translocation  event 
(TAP),  and  the  peptide  selection  and  presenta-
tion event (MHC). New biochemical assays were 
used  to  generate  data  representing  the  above 
events. Significant progress in generating a new 
and  improved  method  for  the  generation  of 
high-density peptide arrays was also made, and 
the researchers hope to implement this technol-
ogy in the generation of orders of magnitude for 
more biochemical data in the future. 

Genomes  To  Vaccines  believes  that  the  inte-
gration  of  experiments  and  bioinformatics  will 
lead  to  an  overall  tool-generation  process  that 
requires  fewer  and  more  pointed  experiments, 
with  more  accurate  predictions.  The  resulting 
predictors  will  be  able  to  computationally  (i.e. 
rapidly  and  exhaustively)  translate  and  evalu-
ate  genomic data for the presence of immuno-
genic epitopes. These resources will be hosted at 
the Genomes To Vaccines website and be made 
publicly available. This will enable any interested 
basic or clinical scientist to perform searches for 
immune targets from any microbial organism, or 
tumour, of their choice. 

The researchers expect that this tool will become 
an essential part of medicine in the future, solv-

The data obtained so far has been used to gen-
erate  bioinformatics  tools  capable  of  predict-
ing  the  outcome  of  these  events  in  silico  and 
to  initiate  the  iterative  process  leading  to  im-
proved predictions. For validation purposes, the 
researchers  have  generated  a  set  of  data  using 
peptides  extracted  from  cellular  MHC,  as  repre-
sentative examples of how the immune system 
works under natural conditions. They integrated 
the prediction tools and demonstrated that such 
an integrated predictor is more successful in pre-
dicting natural epitopes.

Genomes To Vaccines has made these tools gen-
erally  available  as  web-based  services,  and  will 
eventually  also  host  the  data  itself.  Finally,  the 
utility of these tools was demonstrated through 

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IMMUNOTHERAPY ANd TRANSPLANTATION

transport  efficiency,  and  proteasomal  cleavage 
predictions’, Eur J Immunol, 2005, Aug;35(8):2295-
303. 

Sylvester-Hvid, C., Nielsen, M., Lamberth, K., Roder, 
G.,  Justesen,  S.,  Lundegaard,  C., Worning,  P., Tho-
madsen, H., Lund, O, Brunak, S., Buus, S., ‘SARS CTL 
vaccine  candidates;  HLA  supertype-,  genome-
wide scanning and biochemical validation’, Tissue 
Antigens, 2004, May;63(5):395-400. 

Lund,  O.,  Nielsen,  M.,  Kesmir,  C.,  Petersen,  A.G., 
Lundegaard,  C.,  Worning,  P.,  Sylvester-Hvid,  C., 
Lamberth, K., Roder, G., Justesen, S., Buus, S., Bru-
nak,  S.,  ‘Definition  of  supertypes  for  HLA  mol-
ecules  using  clustering  of  specificity  matrices’, 
Immunogenetics, 2004, Mar;55(12):797-810. Epub 
2004 Feb 13. 

Nielsen,  M.,  Lundegaard,  C.,  Worning,  P.,  Hvid, 
C.S.,  Lamberth,  K.,  Buus,  S.,  Brunak,  S.,  Lund,  O., 
‘Improved prediction of MHC class I and class II 
epitopes using a novel Gibbs sampling approach’, 
Bioinformatics,  2004,  Jun  12;20(9):1388-97.  Epub 
2004 Feb 12. 

the prediction of human T cell responses. Initially, 
the researchers showed the speed of these tools 
by  performing  a  complete  epitope  scanning  of 
the SARS virus. 

they  performed  a  complete 
Subsequently, 
epitope  scanning  of  Influenza  A  isolates,  and 
tested the predicted peptides both in biochemi-
cal MHC binding assays, but, more importantly, in 
healthy influenza convalescents. The latter study 
identified  13  new  influenza  epitopes,  which 
covered  virtually  the  entire  population.  These 
epitopes  were  selected  for  being  highly  con-
served,  and  notably,  they  all  encompassed  the 
current bird flu isolates.

The  ability  to  translate  genomic  information 
into immune recognition will form the basis for 
a  rational  approach  to  immunotherapy  (includ-
ing  vaccination),  which  the  researchers  believe 
will serve the policy objectives of enhancing the 
quality of life for EU citizens.

Major publications

Wang,  M.,  Lamberth,  K.,  Harndahl,  M.,  Roder,  G., 
Stryhn,  A.,  Larsen,  M.V.,  Nielsen,  M.,  Lundegaard, 
C.,  Tang,  S.T.,  Dziegiel,  M.H.,  Rosenkvist,  J.,  Ped-
ersen, A.E., Buus, S., Claesson, M.H., Lund, O., ‘CTL 
epitopes for influenza A including the H5N1 bird 
flu;  genome-,  pathogen-,  and  HLA-wide  screen-
ing’, Vaccine, 2006, Dec 29; [Epub ahead of print] 

Peters, B., Bui, H.H., Frankild, S., Nielson, M., Lunde-
gaard, C., Kostem, E., Basch, D., Lamberth, K., Harn-
dahl, M., Fleri, W., Wilson, S.S., Sidney, J., Lund, O., 
Buus, S., Sette, A., ‘A community resource bench-
marking predictions of peptide binding to MHC-I 
molecules’, PLoS Comput Biol, 2006, Jun 9;2(6):e65. 
Epub 2006 Jun 9. 

Larsen,  M.V.,  Lundegaard,  C.,  Lamberth,  K.,  Buus, 
S., Brunak, S., Lund, O., Nielsen, M., ‘An integrative 
approach to CTL epitope prediction: a combined 
algorithm  integrating  MHC  class  I  binding,  TAP 

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gENOMES TO vACCINES

IMMUNOTHERAPY ANd TRANSPLANTATION

Coordinator 

SørenBuus
University of Copenhagen
Institute of Medical Microbiology and Immunology
Panum 18.3.12
Blegdamsvej 3
DK-2200 Copenhagen N, Denmark
E-mail: sb@immi.ku.dk 

Partners

StefanStevanovic
University of Tübingen
Tübingen, Germany

PetervanEndert
INSERM U25
Hôpital Necker
Paris, France

HansjörgSchild
University of Mainz
Mainz, Germany

SørenBrunak
Technical University of Denmark
Lyngby, Denmark

ClausSchafer-Nielsen
Symbion Science Park
Copenhagen, Denmark

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IMMUNOTHERAPY ANd TRANSPLANTATION
COMPUvAC

Rationaldesignandstandardisedevaluationofgeneticvaccines






ContractNo 
Projecttype

LSHB-CT-2004-00246
IntegratedProject

ECcontribution  e 7969442

Startingdate 1January200
Duration 48months

Website  www.compuvac.org

Background and objectives:

CompuVac  has  assembled  16  European  teams 
from both academia and industry, in 9 European 
countries, with multidisciplinary expertise in the 
fields  of  vaccine  development,  immunology,  vi-
rology, vectorology, biomathematics and compu-
ter sciences. Recombinant viral vectors and virus-
like particles are considered the most promising 
vehicles to deliver antigens in prophylactic  and 
therapeutic vaccines against infectious diseases 
and cancer. Several potential vaccine designs ex-
ist,  but  their  cost-effective  development  cruelly 
lacks a standardised evaluation system.

CompuVac  is  devoted  to  the  standardisation  of 
vaccine evaluation and the rational development 
of a novel platform of recombinant vaccines, with 
HCV as a target disease. The CompuVac consor-
tium has several primary objectives:

• 
to  standardise  the  qualitative  and 
quantitative  evaluation  of  genetic  vac-
cines  using  defined  ‘gold  standard’  anti-
gens and methods;
• 
to  rationally  develop  a  platform  of 
novel  genetic  vaccines  using  genomic 
and proteomic information, together with 
the project’s gold standards;
• 
to generate and make available to the 
scientific  community  a ‘tool  box’  and  an 
‘interactive database’ allowing for a com-
parative  assessment  of  future  vaccines 
to  be  developed  with  the  project’s  gold 
standards.

CompuVac members : The CompuVac 2nd Annual 
Meeting, November 2006, Athens, Greece

Approach and methodology:

As  end  products,  the  consortium’s  vector  plat-
form  and  ‘gold  standard’  tools,  methods  and 
algorithms  will  be  made  available  to  the  scien-
tific and industrial communities as a toolbox and 
interactive  database.  CompuVac  seeks  to  apply 
these vectors, tools and methods to the develop-
ment of a preventive and/or therapeutic vaccine 
against  Hepatitis  C  Virus,  incorporating  one  or 
more of the project’s platform vectors expressing 
the HCV envelope protein.

CompuVac  recognises  that  a  uniform  method 
for side-by-side qualitative and quantitative vac-
cine  evaluation  is  lacking.  Thus,  standardising 
the  evaluation  of  vaccine  efficacy  and  molecu-
lar  signature  by  using ‘gold  standard’  tools,  and 
molecular and cellular methods in virology and 
immunology, as well as algorithms based on ge-
nomic information, is key.

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IMMUNOTHERAPY ANd TRANSPLANTATION

Main findings:

Within the first 2 years of its development, 4 ma-
jor  platforms  have  been  set  up  by  CompuVac. 
These are presented below:

1.The CompuVac vector provider platform. This 
platform is dedicated to the engineering and/or 
optimisation  of  vectors  expressing  and/or  con-
taining  the  chosen  model  epitopes.  A  rational 
vector design is carried out based on up-to-date 
genomic  and  proteomic  information.  This  plat-
form assembles a wide range of viral vectors and 
virus-like particles that are among today’s most 
promising vaccine candidates:
•inertparticlevaccines—antigendisplay:

i.  VLPs derived from retrovirus, polyoma-
viruses, hepatitis B virus, bacteriophage,
ii.  Polyepitope peptides;

• gene expression vectors — antigen/trans-
gene/VLPexpression:

i.  Adenovirus/adeno-VLP,
ii.  Herpes Simplex Virus 1/HSV-VLP,

iii.  Measles virus/Measles-VLP,
iv.  MVA/MVA-VLP,
v.  Polyoma virus;

•BCG;
•DNA/DNA-VLP.

There are two selected ‘gold standard’ antigens:

•  T-cell  antigen:  GP33-41  epitope  from 
LCMV,  to  study  the  T  cell  response.  The 
LCMV  glycoprotein  G1  peptide  p33-41  is 
an  extremely  well-characterised  model 
antigen that has been widely used in nu-
merous  studies  for  evaluating  humoral 
response. In addition, this is a virus that is 
mainly  controlled  by  cellular  immune  re-
sponses in vivo.
•  B-cell antigen: VSV-G protein, to study 
the B cell response. The VSV envelop pro-
tein is the viral envelop that is best pseu-
dotyped  on  retroviral-based  particles 
used for neutralisation assays. In addition, 
this is a virus that is mainly controlled by 
humoral immune responses in vivo.

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IMMUNOTHERAPY ANd TRANSPLANTATION

These ‘gold standard’ antigens have been intro-
duced in the set of vectors of CompuVac.

2. The  CompuVac  immunological  testing  plat-
form.  Once  produced,  the  immunological  re-
sponse  will  be  studied  by  the  immunological 
testing platform regarding T and B cell response 
and molecular signature. Data have been gener-
ated and entered in the ‘Genetic Vaccine Decision 
Support system’ (GeVaDSs). ‘Gold standard’ algo-
rithms  for  the  intelligent  interpretation  of  vac-
cine  efficacy  and  molecular  signature  are  built 
into  CompuVac’s  interactive    GeVaDSs,  which 
generates the following: 

•  vector  classification  according  to  in-
duced immune response quality, account-
ing for gender and age;
•  vector combination counsel for prime-
boost immunisations;
•  vector  molecular  signature  according 
to genomic analysis.

A  GeVaDSs  user  manual  for  the  current  version 

Fig 1 & 2: Radar and Bar charts - CompuVac, Genetic 
Vaccines Decision Support System : Results

of the system has already been created. Scientific 
forms for immunisation protocols, animal models 
and T cell responses were created, and the immu-
nisation  protocols  were  then  analysed  and  im-
plemented in GeVaDSs. A public test of GeVaDSs 
took place at the CompuVac’s second executive 
committee meeting in Stockholm in June 2006.
There were 2 reports generated by GeVaDSs:

•  The  T  cell  response  project  report:  in 
this  project,  CompuVac  compared  the 
following:  (i)  interferon  g  production,  (ii) 
memory phenotype induction, (iii) clonal 
expansion,  (iv)  peak  of  clonal  response, 
and (v) cytotoxic T lymphocytes response;
•  The molecular signature report.

3. A CompuVac training and dissemination plat-
form. The  exchange  programme  was  effectively 
launched, allowing CompuVac’s young scientists 
to learn new technologies and benefit from the 
partners’ expertise. An expert immunologist and 

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COMPUvAC

mathematician were hired, to train and connect 
the scientists involved in the building-up of Ge-
VaDSs. 

Four  training  sessions  dedicated  to  the  use  of 
GeVaDSs by the data producers and the Compu-
Vac team leaders, were coordinated by the com-
puter scientists in order to improve data integra-
tion into GeVaDSs. A dissemination meeting was 
organised by the CompuVac management team 
at the 2006 annual meeting of the European So-
ciety of Gene Therapy on ‘Perspectives in Vaccine 
Development’. 

The  CompuVac  tool  for  dissemination  is  a  26-
minute  film  dedicated  to  the  project  and  new 
vaccine  development.  A  film  team  (Actes&Avril 
Productions)  was  contracted  for  the  CompVac 
film,  and  sequences  were  shot  between 
November and December 2006 in the following 
locations: 

Institute,  Athens, 
•  Hellenic  Pasteur 
Greece (CompuVac annual meeting, ESGT 
dissemination  meeting,  and  participating 
institute/immunological testing platform);
•  University of Munich, Munich, Germa-
ny (participating institute/immunological 
testing platform);
•  University  of  Zürich,  Zürich,  Switzer-
land (participating institute/immunologi-
cal testing platform);
•  CNRS – Pierre et Marie Curie University, 
Paris, France (participating institute/scien-
tific coordination/management platform);
•  Poznan  University  of  Technology, 
Poznan,  Poland  (participating  institute/
GeVaDSs platform);
•  Cytos Biotec, Zürich, Switzerland (par-
ticipating biotechnology company/vector 
platform),
•  Epixis  SA,  Lyon,  France,  (participating 
biotechnology company/vector platform).

management of the scientists, the administrative 
and  financial  management,  and  the  intellectual 
property management, with a major feature be-
ing the study of the future of GaVaDSs.

Major publications 

Bellier, B., Dalba. C., Clerc, B., Desjardins, D., Drury, 
R., Cosset, F.L., Collins, M., Klatzmann, D., ‘DNA vac-
cines encoding retrovirus-based virus-like parti-
cles induce efficient immune responses without 
adjuvant’, Vaccine, 2006, Mar 24;24(14):2643-55.

Dalba,  C.,  Bellier,  B.,  Kasahara,  N.,  Klatzmann,  D., 
‘Replication-competent  Vectors  and  Empty  Vi-
rus-like  Particles:  New  Retroviral Vector  Designs 
for  Cancer  Gene Therapy  or Vaccines’,  Mol. Ther, 
2007, Jan 23.

Dreux, M., Pietschmann, T., Granier, C., Voisset, C., 
Ricard-Blum, S., Mangeot, P.E., Keck, Z., Foung, S., 
Vu-Dac, N., Dubuisson, J., Bartenschlager, R., Lavil-
lette,  D.,  Cosset,  F.L.,  ‘High  density  lipoprotein 
inhibits  hepatitis  C  virus-neutralizing  antibod-
ies  by  stimulating  cell  entry  via  activation  of 
the scavenger receptor BI’ J Biol Chem, 2006, Jul 
7;281(27):18285-95.

Tegerstedt,  K.,  Franzen,  A.,  Ramqvist,  T.,  Dalianis, 
T., ‘Dendritic cells loaded with polyomavirus VP1/
VP2Her2  virus-like  particles  (VLPs)  efficiently 
prevent  outgrowth  of  a  Her2/neu  expressing 
tumour’,  Cancer  Immunology  Immuntherapy,  In 
press, 2007.

Sominskaya, 
I.,  Alekseeva,  E.,  Skrastina,  D., 
Mokhonov,  V.,  Starodubova,  E.,  Jansons,  J.,  Levi, 
M., Prilipov, A., Kozlovska, T., Smirnov, V., Pumpens, 
P.,  Isaguliants,  M.G.,  ‘Signal  sequences  modu-
late  the  immunogenic  performance  of  human 
hepatitis  C  virus  E2  gene’,  Mol  Immunol,  2006, 
May;43(12):1941-1952.

4. The  CompuVac  management  platform  has 
been set up, and is dedicated to the day-to-day 

Bartosch, B., Cosset, F.L., ‘Cell entry of hepatitis C 
Virus’, Virology, 2006, Apr 25;348(1):1-12.

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IMMUNOTHERAPY ANd TRANSPLANTATION

CharlotteDalba
Epixis SA
Paris, France

AlbertoEpstein
Université Claude Bernard Lyon 1
Villeurbanne, France

StefanKochanek
Universitätsklinikum Ulm
Ulm, Germany

PenelopeMavromara
Hellenic Pasteur Institute
Athens, Greece

AlbertusOsterhaus
Erasmus MC Rotterdam
Rotterdam, Netherlands

PaulPumpens
BioMedical Research and Study Centre
Riga, Latvia

KestutisSasnauskas
Institute of Biotechnology
Vilnius, Lithuania

RudolfMartinZinkernagel
University of Zurich
Institute of Experimental Immunology
Zurich, Switzerland

Lavillette,  D.,  Bartosch,  B.,  Nourrisson,  D., Verney, 
G., Cosset, F.L., Penin, F., Pecheur, E.I., ‘Hepatitis C 
virus  glycoproteins  mediate  low  pH-dependent 
membrane  fusion  with  liposomes’,  J  Biol  Chem, 
2006, Feb 17;281(7):3909-17.

Coordinator 

DavidKlatzmann
CNRS and Université Pierre et Marie Curie
Laboratoire de Biologie et Thérapeutique des Pathogies 
Immunitaires
UMR 7087 – UPMC-CNRS
Groupe Hospitalier Pitié-Salpêtrière
Bat. CERVI, 83, boulevard de l’Hôpital
75651 Paris Cedex 13 - France
E-mail : david.klatzmann@chups.jussieu.fr

Partners 

AnatolySharipo
ASLA Biotech Ltd
Riga, Latvia

JacekBlazewicz
Poznan University of Technology
Poznan, Poland

ThomasBrocker
Institute of Immunology - Ludwig-Maximilians-Univerität 
München
Munich, Germany

François-LoïcCosset
INSERM
Lyon, France

GaryThomasJennings
Cytos Biotechnology AG
Zurich-Schlieren, Switzerland

TinaDalianis
Karolinska Institutet
Stockholm, Sweden

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18

HEPACIvAC

New preventative and therapeutic Hepatitis C vaccines: from preclinical to
phaseI

IMMUNOTHERAPY ANd TRANSPLANTATION

ContractNo 
Projecttype

LSHB-CT-2007-03743
IntegratedProject

ECcontribution  e 8800000

Startingdate 1January2007
Duration 60months






Background and objectives: 

Liver  disease  caused  by  Hepatitis  C Virus  (HCV) 
infection  is  a  major  medical  problem,  affecting 
an estimated 123 million people worldwide. No 
vaccine is available and the best antiviral therapy 
— a combination of interferon alpha and ribavi-
rin  —  is  only  effective  in  a  minority  of  patients. 
An  increasing  body  of  data  suggests  that  vi-
rus-specific T cell responses are associated with 
clearance of HCV in acutely infected humans and 
chimpanzees. 

Based  on  these  observations,  the  long-term 
objective  of  the  HCV  vaccine  programme  is 
to  develop  both  prophylactic  and  therapeutic 
vaccines that elicit antiviral CD4+ and CD8+ re-
sponses capable of one of the following: 

reducing the rate of incidence and/or 
• 
persistence of HCV infection following ex-
posure (for prophylactic vaccine); 
• 
increasing  the  rate  of  clearance  of 
HCV  infection  as  a  monotherapy,  and/or 
in  combination  with  current  therapy  or 
novel  anti-viral  therapy  (for  therapeutic 
vaccine). 

Approach and methodology: 

The HEPACIVAC project is being carried out by a 
large consortium, which includes two major vac-
cine  manufacturers,  and  several  academic  and 
research  groups  with  expertise  in  clinical  vac-
cine development, human immunity and animal 

models. The consortium will focus on the skills of 
the individual participants towards the develop-
ment of new preventative and therapeutic strat-
egies against HCV infection, integrating different 
approaches and disciplines. 

The HEPACIVAC proposed work is focused on the 
development  of  two  HCV  vaccine  candidates, 
independently  identified  by  IRBM  (Istituto  di 
Ricerche  di  Biologia  Molecolare)  and  Novartis. 
This study will be performed with a strong com-
mitment  to  translate  the  results  into  effective 
approaches  for  the  prevention  and  therapy  of 
HCV.  The  first  vaccine  candidate  is  gene-based, 
encodes for the 2 000 amino acid-long HCV Non 
Structural  region  (from  NS3  to  NS5B)  and  uti-
lises  adenoviral  vectors  for  delivery.  These  vec-
tors have been shown to elicit potent CD4+ and 
CD8+ T cell responses in rodents and primates. 

Recently, a proof-of-concept vaccination and het-
erologous challenge experiment in chimpanzees 
showed that potent, broad and long-lived T-cell 
responses to HCV were elicited in vaccinated ani-
mals. The gene-based vaccine protected against 
acute and chronic disease induced by challenge 
with a high dose of an heterologous HCV strain. 
The  second  vaccine  candidate  consists  of  re-
combinant HCV glycoproteins, gpE1- and gpE2-
associated,  to  resemble  a  pre-virion  envelope 
structure.  Protection  against  homologous  and 
heterologous challenge, mediated by CD4+ T cell 
response and antibodies, was observed in exper-
iments in chimpanzees.

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IMMUNOTHERAPY ANd TRANSPLANTATION

•  demonstration  of  immunogenicity  of 
the  gene-based  vaccine,  in  HCV  chroni-
cally infected chimpanzees;
•  demonstration  of  immunogenicity  of 
the two vaccines administered in combi-
nation in macaques;
• 
assays;
•  evaluation  of  safety  and  immuno-
genicity of each of the two candidate vac-
cines, in healthy volunteers and in chroni-
cally infected individuals.

standardisation of the immunological 

It follows that both vaccine candidates have the 
potential to protect humans from a large number 
of viral strains. The complementary action of these 
vaccines might be highly instrumental in making 
a vaccine against HCV, which is able to stimulate 
several components of the immune system and 
to  elicit  efficacious  immune  responses,  both  in 
preventative  and  therapeutic  vaccination  set-
tings. Furthermore, since the two vaccines target 
different arms of the acquired immune response, 
HEPACIVAC will offer the opportunity to analyse 
in  detail  immune  correlates  of  protection  from 
HCV infection, as well as to standardise immuno-
genicity assays, and to establish benchmark ref-
erences for future preclinical and clinical studies 
of HCV candidate vaccines.

During the first part of the project and after the 
production of GLP & GMP vaccines, the work will 
focus  on  several  areas:  animal  safety  and  toler-
ability studies for the gene-based vaccine; and a 
preclinical  evaluation  of  compatibility,  synergis-
tic  or  antagonistic  actions  of  the  two  vaccines 
administered in combination in macaques. These 
studies, aimed at verifying the possibility of using 
this vaccine in humans, will be accompanied by 
specific activities on preclinical and clinical sam-
ples, in order to identify and standardise suitable 
immunological  assays  to  be  used  in  the  follow-
ing HCV vaccine clinical trials. 

In  the  second  part  of  the  project,  HEPACIVAC 
plans to perform Phase I trials with the vaccines 
separately, to assess safety, tolerability and immu-
nogenicity  in  healthy  volunteers  and  in  chroni-
cally infected individuals.

Expected outcome:

HEPACIVAC anticipates the following results:

•  production of vaccine lots in sufficient 
amounts to conduct preclinical and clini-
cal studies; demonstration of safety in ani-
mals, of the gene-based vaccine;

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IMMUNOTHERAPY ANd TRANSPLANTATION

KrystinaBienkowska-Szewczyk
University of Gdansk
Gdansk, Poland

SayedF.Abdelwahab
The Egyptian Company for Diagnostics
Agouza Giza, Egypt
Cristiana Tozzi
ALTA Srl
Siena, Italy

AdrianHill
University of Oxford
Oxford, UK

HEPACIvAC

Coordinator

RiccardoCortese
CeInge Biotecnologie avanzate s.c.a.r.l.
via Pansini 5
80131 Naples, Italy
E-mail: cortese@ceinge.unina.it

Scientific coordinator

SergioAbrignani
Novartis Vaccine & Diagnostics
via Fiorentina 1
53100 Siena, Italy
E-mail: sergio_abrignani@chiron.com

Partners

AlfredoNicosia
Istituto di Ricerche di Biologia Molecolare (IRBM) 
P.Angeletti
Pomezia (RM), Italy

FerruccioBonino
Fondazione IRCCS Ospedale Maggiore Mangiagalli e 
Regina Elena
Milan, Italy

AlbertD.M.E.Osterhaus
Erasmus Medical Centre
Rotterdam, Netherlands

GeertLerouxRoels
University of Ghent
Ghent, Belgium

JaneMcKeating
University of Birmingham
Birmingham, UK

StefanZeuzem
University of Saarland
Saarland, Germany

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IMMUNOTHERAPY ANd TRANSPLANTATION
BACABS

Assessment of structural requirements in complement-mediated bactericidal
events:‘Towardsaglobalapproachtotheselectionofnewvaccinecandidates’






ContractNo 
Projecttype

LSHB-CT-2006-03732
SME-SpecificTargetedResearchProject

ECcontribution  e 2269999

Startingdate 1January2007
Duration 36months

Website  www.bacabs.org

Background and objectives:

High throughput cloning and expression of large 
sets of genomic open reading frames (ORFs) has 
become the preferred industrial strategy for ge-
nome-wide searches of new vaccine candidates. 
For  invasive  infections  in  particular,  the  aim  is 
to  find  proteins  eliciting  antibodies  capable  of 
binding to the bacterial cell surface, and through 
interaction  with  the  complement  system  effec-
tively killing the bacteria. 

However, current data from reverse vaccinology 
studies  (the  targeting  of  possible  vaccine  can-
didates,  beginning  with  genomic  information) 
show  that  only  a  small  fraction  of  surface-ex-
posed proteins appears to elicit antibodies with 
bactericidal  activity.  Using  information  gener-
ated by reverse vaccinology projects within the 
consortium, the BacAbs project will apply a novel 
multidisciplinary approach that will help to sin-
gle  out  the  structural  requirements  for  viable 
bactericidal vaccine candidates, and will develop 
bioinformatics tools to predict compliance with 
such structural requirements. 

To this end, a systematic analysis of the sequence, 
structure, dynamics and interactions of selected 
protein  targets  will  be  conducted,  using  the 
serogroup-B  Neisseria  meningitidis  as  a  model 
system. The consortium consists of an industrial 
partner  with  extensive  experience  in  vaccine 
development,  three  SMEs  with  strong  expertise 
in  several  key  technological  aspects  of  the 

project,  and  five  academic  partners  that  are 
internationally recognised for their experimental 
and theoretical studies of protein structure and 
interactions.

The development of antibiotic resistance consti-
tutes one of the most potentially serious threats 
in modern medicine. One approach to minimis-
ing the use of antibiotics, is to vaccinate against 
pathogenic strains of bacteria. A clear candidate 
for this approach is Neisseria meningitidis, a ma-
jor  cause  of  bacterial  septicemia  and  meningi-
tis  against  which  no  effective  vaccine  exists.  N. 
meningitidis  is  a  Gram-negative  bacterium  that 
is  capsulated  in  its  invasive  form,  and  classified 
into  five  major  pathogenic  serogroups  on  the 
basis of the chemical composition of distinctive 
capsular polysaccharides. Although a promising 
candidate  has  entered  clinical  development,  no 
effective vaccine against serogroup-B N. menin-
gitidis (MenB), which is responsible for more than 
50% of all meningococcal diseases in Europe, has  
reached  the  market  yet.  The  capsular  polysac-
charide  of  MenB  is  identical  to  that  of  a  widely 
distributed human carbohydrate, making its use 
as the basis of a vaccine for prevention of MenB 
diseases problematic. Consequently, most efforts 
have  turned  to  the  development  of  vaccines 
based on surface-exposed or exported proteins.

A  bactericidal  response,  i.e.  one  that  leads  to 
bacterial-cell death, can be triggered through a 
variety of methods. For meningococcal infections, 
in  vitro  bactericidity  assays  with  immune  sera, 

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IMMUNOTHERAPY ANd TRANSPLANTATION

BACABS

correlate  with  protection  in  humans.  Although 
in  vivo  protection  against  MenB  may  not  be 
solely  achieved  by  complement-dependent 
bacteriolysis,  an  antigen  that  elicits  (murine) 
antibodies  capable  of  triggering  bacterial-cell 
in  a  complement-dependent 
death 
manner,  is  normally  considered  a  candidate  for 
human vaccine development.

in  vitro 

In this context, one major obstacle to vaccine de-
velopment, besides sequence and antigenic vari-
ability,  is  the  difficulty  to  identify  antigens  that 
will  generate  a  bactericidal  response.  Typically, 
only a very small fraction of the antibodies raised 
in large-scale antigen-screening studies are bac-
tericidal.  Thus,  while  potential  antigens  can  be 
readily identified, this information is of little use 
unless  we  are  also  able  to  predict  the  antigens 
that  can  lead  to  the  production  of  bactericidal 
antibodies.

In principle, the capacity of a protein antigen to 
raise  bactericidal  antibodies  may  depend  on  (a 
combination of ) the following factors: 

• 
size,  shape,  structural  complexity, 
abundance, solubility, propensity and oli-
gomerisation; 
• 
location  of  specific  protein 
(epitopes). 

sequence,  structure,  dynamics  and 
regions 

These factors may in turn modulate the properties 
of the Ag-Ab complex and its susceptibility to be 
recognised by the C1q component. The BacAbs 
project aims at deciphering possible correlations 
between these factors and bactericidity.

Approach and methodology:

Following  the  framework  outlined  above,  the 
BacAbs  project  is  concerned  with  the  identifi-
cation of (surface-exposed or exported) protein 
antigens  that  may  elicit  complement-mediated 
bactericidity in vitro. This includes early discrimi-
nation  of  antigens  that  may  induce  production 

of non-bactericidal antibodies. 

To  achieve  this  goal,  the  consortium  will  inves-
tigate  the  requirements  for  a  productive  Ag-
Ab-C1q  complex  formation.  It  proposes  to  find 
relevant  answers  using  a  multidisciplinary  and 
comparative approach to study the structure of 
a number of these complexes. The MenB vaccine 
development  project  of  Novartis Vaccines  &  Di-
agnostics is to serve as a model, while a source 
of  useful  data  and  reagent  molecules  will  be 
employed  as  well.  To  single  out  possible  struc-
tural determinants, the focus is being placed on 
groups of antigens with similar size, abundance, 
solubility,  etc.  (both  eliciting  and  not  eliciting 
bactericidal antibodies).

Although  initially  centred  around  group-B  N. 
meningitidis,  the  specific  target  of  the  project 
is the development of tools that can be applied 
effectively to the genome-wide identification of 
vaccine candidates against any bacterial patho-
gen susceptible to complement-mediated lysis.

RoleofSMEs

The  three  SMEs  involved  in  the  BacAbs  project 
have  a  principal  technological  role. The  IT  com-
pany INFOCIENCIA SL will be operating the man-
agement  and  dissemination  web  servers  of  the 
Consortium, carrying out bioinformatics analysis 
of antigen and epitope sequences, implementing 
algorithms,  protocols  and  data  emerging  from 
the Consortium’s work into computational tools 
and databases within a web-based technological 
platform, and evaluating the commercial interest 
of this platform via demonstration. 

Two biotechs will be working on sample prepara-
tion  and  protein-structure  determination.  ASLA 
Biotech  Ltd  will  be  performing  protein  expres-
sion and labelling; screening and optimisation of 
sample conditions  for NMR  analysis;  monoclonal 
antibody  generation;  and  sequential  backbone 
assignment and structure determination via NMR. 

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IMMUNOTHERAPY ANd TRANSPLANTATION

Potential applications:

The results of the BacAbs project could eventu-
ally  be  applied  in  the  selection  of  new  vaccine 
candidates  against  group-B  Neisseria  meningi-
tidis and other bacterial pathogens.

Bio-Xtal  SA  will  be  performing  protein  expres-
sion  and  purification,  especially  in  connection 
with  derivatives  (seleno-methionine  labelled), 
for  X-ray  analysis,  expression  and  solubilisation 
screens on highly hydrophobic or poorly soluble 
targets. The consortium will also explore protein 
crystallogenesis  using  nanodrop  scale  robot-
ics techniques based on commercially available 
and  proprietary  screening  approaches;  develop 
and optimise robotic processes for crystallisation 
plate storage and drop visualization; refine crys-
tal  growth  conditions  for  selected  hits  to  yield 
diffraction quality crystals; and collect X-ray data 
and structure solutions.

Expected outcome:

The BacAbs project will provide the following:

•  Structural information on a set of pro-
teins that are components of the cell sur-
face  (the  bacterial  organ  for  interaction 
with eukaryotic host cells) of a major hu-
man pathogen;
• 
Improved experimental protocols and 
techniques,  bioinformatics  tools  and  da-
tabases to assist the development of vac-
cines against human bacterial pathogens 
in general, and group-B Neisseria menin-
gitidis in particular;
•  A  framework  in  which  experimental 
and  in  silico  methods  for  determining 
protein  structure  and  studying  macro-
molecular recognition, immunological re-
sponse mechanisms, and sequence-struc-
ture-function relationships can be further 
developed;
•  A  web-based  technological  platform 
integrating  this  knowledge,  with  the  po-
tential  to  improve  the  effectiveness  and 
reducing  the  costs  of  vaccine-candidate 
searches.

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IMMUNOTHERAPY ANd TRANSPLANTATION

BACABS

Coordinator

XavierDaura
Universitat Autònoma de Barcelona Campus UAB s/n
Institución Catalana de Investigaciòn y
 Estudios Avanzados
08193 Bellaterra (Cerdanyola del Vallès), Spain
E-mail: Xavier.Daura@uab.es

Partners

GuidoGrandi
Novartis Vaccines and Diagnostics
Siena, Italy

AnatolySharipo
ASLA BIOTECH, Ltd.
Riga, Latvia

GhericiHassaine
Bio-Xtal SA
Mundolsheim, France

GiorgioColombo
Consiglio Nazionale delle Ricerche
Istituto di Chimica del Riconoscimento Molecolare
Milan, Italy

MartinZacharias
International University Bremen GmbH
School of Engineering and Science
Bremen, Germany

MartinoBolognesi
Università degli Studi di Milano
Milan, Italy

AlexandreM.J.J.Bonvin
Universiteit Utrecht
Department of Chemistry
Utrecht, Netherlands

JoséManuelMasBenavente
INFOCIENCIA SL
Barcelona, Spain

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IMMUNOTHERAPY ANd TRANSPLANTATION
MIMOvAX

Alzheimer’s disease-treatment targeting truncated Aß 40/42 by active
immunisation






ContractNo 
Projecttype

LSHB-CT-2006-037702
SME-SpecificTargetedResearchProject

ECcontribution  e 23701

Startingdate 1October2006
Duration 36months

Website  www.mimovax.eu

Background and objectives:

Alzheimer’s  disease  (AD)  is  the  most  common 
form of dementia in humans. The Alzheimer As-
sociation  has  observed  that  there  are  currently 
12 million patients worldwide suffering from this 
disease, with estimated social costs for every pa-
tient  reaching  Δ40  000  per  year.  Currently,  there 
is  no  effective  treatment  available  to  stop  the 
progressive  neurodegeneration  and  associated 
cognitive decline in human patients. The current 
cases are expected to almost double within the 
next 20 years. All the treatment strategies pres-
ently  being  applied  are  focusing  on  the  use  of 
small  molecular  drugs  that  inhibit  the  activity 
of cholinesterase, to alleviate disease symptoms. 
These drugs, however, have not been proven to 
effectively  halt  or  to  revert  disease  progression 
after prolonged treatment. 

Alzheimer’s disease is caused by the deposition 
of  Beta-Amyloide  (BA)  into  plaques  within  the 
patients’ brain. The main constituent of BA is pro-
tein fragments, of 40-42 amino acids. These frag-
ments derive from the Amyloid Precursor Protein 
(APP), which is expressed on various cell types in 
the  nervous  system.  In  humans,  the  majority  of 
amyloid  plaque  material  is  formed  by  Aß40/42 
derivatives  which  are  frequently  truncated  and 
modified.  Aß  peptides  are  considered  to  be  di-
rectly involved in the pathogenesis and progres-
sion of AD. 

Consequently,  reduction  of  Aß  burden  in  the 

brain  is  predicted  to  slow  down  or  halt  disease 
progression,  and  could  also  stop  cognitive  de-
cline in AD patients.

Indeed, 
immunotherapeutic  treatment  using 
active  and  passive  immunisation  strategies  to 
target full length Aß led to the reduction of Aß 
plaques and had a beneficial impact on disease 
progression  in  animal  models  of  AD.  However, 
the first phase II vaccination trial in AD patients 
using  full  length  Aß42  as  an  antigen  had  to  be 
discontinued, due to severe neuroinflammatory 
side effects, including brain infiltration by auto-
reactive T cells. 

MimoVax — Alzheimer’s vaccine — is a specific 
targeted research project (STREP) for the devel-
opment and optimisation of a first treatment to 
stop the progression of AD. This project aims at 
developing a vaccine against modified forms of 
BA. The immune system of AD patients will be ac-
tivated to attack and remove BA, and will be able 
to fight the cause of the disease directly. 

The innovative technology presented in MimoVax 
has been developed to create antigens mimicking 
the structure of neo-epitopes which do not con-
tain sequences of the native Aß peptide. A Mimo-
tope-based  AD  vaccine  would  induce  antibody 
responses, exclusively reacting with the patholog-
ical Aß molecules but not with parental structures 
like APP. Furthermore, Mimotopes do not contain 
potential T cell self-epitopes and they avoid induc-
tion of detrimental autoreactive T cells. 

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IMMUNOTHERAPY ANd TRANSPLANTATION

didates will be tested for efficacy in animal mod-
els of AD, altering disease progression. 

This evaluation will include analysis of AD-pathol-
ogy in the brain, as well as the testing of memory 
and learning in these animals by cognitive tests 
in  vivo.  The  successful  candidates  will  then  be 
tested for tolerability and safety in a first phase 
I trial in AD patients. Additionally, new diagnos-
tic methods will be developed and evaluated in 
order to monitor treatment efficacy in vivo in ani-
mal models.

Expected outcome:

The expected outcome of the project will com-
prise a proof of concept for active immunisation 
using MimoVax vaccines in animal models. Suc-
cessful treatment should result in a reduction of 
pathologic alterations in the brain, as  well as in 
improved  learning  and  memory  of  the  treated 
animals. Furthermore, the researchers expect to 
demonstrate  the  safety  and  tolerability  of  the 
vaccine in patients, in initial phase I testing. 

In summary, vaccines based on Mimotope pep-
tides mimicking neo-epitopes which are derived 
from the highly pathological and abundant Amy-
loid-Δ derivatives, provide an innovative, inexpen-
sive  and  efficient  way  to  treat  this  widespread 
disease  with  its  severe  personal  and  economic 
impact on patients, their families and society.

MIMOvAX

Amyloid plaque staining in the brain of an AD mouse. 
One of the hallmarks of Alzheimer’s disease is the accu-
mulation of amyloid plaques in the brain. These amyloid 
plaques can be visualised on sections of these brains by 
staining with amyloid specific antibodies. This staining 
reaction results in a red amyloid plaque (indicated by a 
white arrow) surrounded by blue nuclei of surrounding 
cells like neurons or astrocytes. A Mimotope-based AD 
vaccine as described in this abstract would induce such 
specific antibody responses reacting with the pathologi-
cal AΔ molecules and could therefore be a safe treatment 
regimen to efficiently fight AD in patients.

Despite  the  fact  that  similar  to  full  length  Aß, 
truncated and modified Aß peptides seem to be 
involved in the pathogenesis and progression of 
AD,  no  relevant  development  programme  has 
been  initiated  to  date.  The  goal  of  MimoVax  is 
the development of a safe and efficacious Alzhe-
imer  vaccine,  which  can  prevent  or  even  revert 
cognitive decline in AD patients. In addition, new 
diagnostic  methods  will  be  developed  in  order 
to monitor treatment efficacy.

Approach and methodology:

This novel vaccine-technology is based on Mim-
otope  peptides  mimicking  neo-epitopes  which 
are  present  on  the  above-mentioned  highly 
pathological  and  abundant  Amyloid-ß  deriva-
tives, but not on the parental Amyloid Precursor 
Protein (APP). The first step in this project will be 
the identification and evaluation of novel mimo-
topes  mimicking  epitopes  present  on  different 
Aß derivatives. Subsequently, these vaccine can-

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IMMUNOTHERAPY ANd TRANSPLANTATION

Coordinator 

FrankMattner
Affiris GmbH
Campus Vienna Biocentre
Viehmarktgasse 2a
A-1030 Vienna, Austria
E-mail: frank.mattner@affiris.com

Scientific coordinator

MarkusMandler
Affiris GmbH
Campus Vienna Biocentre
Viehmarktgasse 2a
A-1030 Vienna, Austria
E-mail: markus.mandler@affiris.com

Partners 

IrisGrünert
Biolution gruenert & co KEG
Vienna, Austria

AntónAlvarez
EUROESPES, SA
Department of Neuropharmacology
A Coruna, Spain

ManfredWindisch
JSW – Research GmbH
Graz, Austria

FritzAndreae
piCHEM research development 
Graz, Austria

RichardDodel
Philipps-Universität Marburg
Marburg, Germany

AlexanderDrzezga
Technische Universität München –
 Klinikum rechts der Isar
Munich, Germany

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19

PHARMA-PLANTA 

Recombinantpharmaceuticalsfromplantsforhumanhealth

IMMUNOTHERAPY ANd TRANSPLANTATION






ContractNo 
Projecttype

LSHB-CT-2003-036
IntegratedProject

ECcontribution  e 12million

Startingdate 1February2004
Duration 60months

Website  www.pharma-planta.org

Background and objectives: 

Pharma-Planta aims to build a plant-based pro-
duction platform for pharmaceuticals in Europe 
and to enter the first candidate pharmaceuticals 
into  clinical  trials.  The  programme  will  develop 
robust  risk-assessment  and  risk-management 
practices based on health and environmental im-
pact and will work with EU regulatory authorities 
to ensure safety and acceptance. 

Plants have enormous potential for the produc-
tion of recombinant pharmaceutical proteins as 
they are inexpensive and versatile, in addition to 
them being amenable to rapid and economical 
scale-up. A major goal will be to address the nec-
essary  biosafety  and  regulatory  requirements 
for  the  use  of  plant-derived  pharmaceuticals 
through a process of engagement and consulta-
tion  with  the  regulatory  bodies  involved  in  GM 
plants, as well as new medicines. The project ad-
dresses  pharmaceuticals  for  the  prevention  of 
HIV,  rabies,  tuberculosis  and  diabetes,  which  re-
main significant health problems, both in Europe 
and the developing world. 

The  Pharma-Planta  consortium  partners  repre-
sent many of the major laboratories in Europe fo-
cusing on the creation of transgenic plants that 
express  important  pharmaceuticals  for  human 
health.  Collectively,  the  consortium  has  a  wide 
range of expertise spanning the areas of molecu-
lar and plant biology, immunology, recombinant 
protein expression technology, vaccinology, plant 

biotechnology,  risk  assessment  and  IP  manage-
ment. This  is  a  unique  opportunity  to  make  an 
impact on EU and global health, through the re-
sponsible development of plant biotechnology.

Approach and methodology: 

The Pharma-Planta project is divided into 6 work 
packages  (WPs),  focusing  on  distinct  aspects  of 
pharmaceutical  production  in  plants.  WP1  ex-
amines targets and is responsible for producing 
expression constructs and assays for the disease 
target proteins to be expressed in plants. In this, 
the  third  year  of  the  programme,  the  role  of 
WP1 is all but complete. WP2 is concerned with 
regulatory  guidance  and  engagement  with  the 
regulatory authorities. WP3 focuses on the major 
production crops — maize and tobacco — which 
were chosen early in the project after consulta-
tions carried out in WP2. 

Of the 8 targets, 2 were chosen in WP1, and were 
‘fast-tracked’  in  WP3,  which  means  they  were 
selected  for  breeding,  scale-up  and  eventual 
production under good manufacturing practice 
(GMP)  conditions.  It  was  decided  at  the  start  of 
the  project  that  these  targets  would  be  2  anti-
HIV antibodies. However, WP3 is concerned only 
with  the  upstream  production  and  the  genera-
tion  of  plant  material  ready  for  extraction  and 
processing of these antibodies. WP3 also looks at 
alternative expression platforms, including plas-
tid  transformation  in  tobacco,  tomato  and  let-
tuce. WP4 is called the development loop, and its 

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IMMUNOTHERAPY ANd TRANSPLANTATION

role is to examine various different strategies to 
improve production yield and product quality. 

but also through a significant number of interac-
tions between WP2 and the regulatory authori-
ties in Europe and South Africa. 

Different  workgroups  within  WP4  are 
look-
ing  at  fusion  proteins  to  enhance  stability,  pro-
tease knockouts, genetic modification of glycan 
structures,  protein  targeting  and  even  the  use 
of plant viruses as expression vectors. Novel dis-
coveries and innovations in WP4 will be benefi-
cial to molecular farming as a whole and will be 
exploited  in  future  programmes. WP5  and WP6 
have gained importance as we enter the second 
half of the project. WP5 is concerned with down-
stream processing – extraction, clarification, pu-
rification and formulation of the antibodies as a 
clinical batch under GMP conditions. WP6, which 
will run until the end of the project, is the clinical 
trial itself. 

Expected outcome: 

The  overall  aim  of  the  project  is  to  complete  a 
phase  I  trial  in  humans,  using  either  or  both  of 
the 2 anti-HIV antibodies selected for fast-track-
ing through production, processing and regula-
tory compliance. At this stage of the project, the 
Pharma-Plant  consortium  remains  convinced 
that this outcome will be achieved.

Main findings: 

In the first year of the project, the 2 anti-HIV anti-
bodies were chosen as fast-track molecules and 
the other 6 targets were consigned to the devel-
opment loop. Another significant decision was to 
focus on maize and tobacco as dual production 
systems.  Pharma-Plant  has  achieved  high  levels 
of antibody expression in both systems, and fully 
expects to achieve a clinical batch of antibody for 
clinical trials by 2009. The project partners have 
achieved significant progress in the area of proc-
ess development, to allow the extraction and pu-
rification of antibody molecules from plant tissue 
under GMP conditions. This has been facilitated 
not  only  by  process  development  work  in WP5, 

Major publications 

Sparrow, P.A.C., Irwin, J.A., Dale, P.J., Twyman, R.M., 
Ma,  J.K.C., ‘Pharma-Planta:  Road  testing  the  de-
veloping  regulatory  guidelines  for  plant-made 
pharmaceuticals’,  Transgenic  Res.,  2007,  Feb  7, 
16(2):147-61. Epub. 

Van Droogenbroeck, B., Cao, J., Stadlmann, J., Alt-
mann,  F.,  Colanesi,  S.,  Hillmer,  S.,  Robinson,  D.G., 
Van  Lerberge,  E.,  Terryn,  N.,  Van  Montagu,  M., 
Liang,  M.,  Depicker,  A.,  Jaeger,  G.D., ‘Aberrant  lo-
calization and underglycosylation of highly accu-
mulating single-chain Fv-Fc antibodies in trans-
genic Arabidopsis seeds’, Proc Natl Acad Sci USA, 
2007, 104(4):1430-1435.

Dunkley,  T.P.,  Hester,  S.,  Shadforth,  I.P.,  Runions, 
J., Weimar, T., Hanton, S.L., Griffin, J.L., Bessant, C., 
Brandizzi, F., Hawes, C., Watson, R.B., Dupree, P., Lil-
ley, K.S. ‘Mapping the Arabidopsis organelle pro-
teome’, Proc Natl Acad Sci USA, 2006, 103(17):6518-
65123. 

Ma,  J.K.,  Barros,  E.,  Bock,  R.,  Christou,  P.,  Dale,  P.J., 
Dix, P.J., Fischer, R., Irwin, J., Mahoney, R., Pezzotti, 
M.,  Schillberg,  S.,  Sparrow,  P.,  Stoger,  E., Twyman, 
R.M.,  ‘European  Union  Framework  6  Pharma-
Planta  Consortium  Molecular  farming  for  new 
drugs and vaccines. Current perspectives on the 
production  of  pharmaceuticals  in  transgenic 
plants. EMBO Rep., 2005, 6(7):593-599.

Ma,  J.K.,  Chikwamba,  R.,  Sparrow,  P.,  Fischer,  R., 
Mahoney,  R., Twyman,  R.M., ‘Plant-derived  phar-
maceuticals - the road forward’, Trends Plant Sci., 
2005, 10(12):580-585. 

Nuttall,  J.,  Ma,  J.K.,  Frigerio,  L., ‘A  functional  an-
tibody  lacking  N-linked  glycans  is  efficiently 
folded,  assembled  and  secreted  by  tobacco 

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IMMUNOTHERAPY ANd TRANSPLANTATION

PHARMA-PLANTA 

mesophyll  protoplasts’,  Plant  Biotechnol  J.,,2005,  
3(5):497-504. 

Twyman, R.M., Schillberg, S., Fischer, R., ‘Transgen-
ic plants in the biopharmaceutical market’, Expert 
Opin Emerg Drugs., 2005, 10(1):185-218.

PhilipJ.DaleandGeorgeLomonossoff
John Innes Centre
Norwich, UK

LaurenceDedieuandRogerFrutos
Centre de Coopération Internationale en Recherche 
Agronomique pour le Développement (CIRAD) 
Paris, France

Coordinator 

RainerFischer
Fraunhofer Gesellschaft
Hansastrasse 27c
D-80686 Munich, Germany
E-mail: fishcer@molbiotech-aachen.de

Scientific coordinator

JulianMa
St. George’s Hospital Medical School
Department of Cellular and Molecular Medicine (CMM)
Cranmer Terrace
London SW17 0RE, UK
E-mail: jma@sghms.ac.uk 

ChrisHawes
Oxford Brookes University
Oxford, UK

LorenzoFrigerio
University of Warwick
Coventry, UK

FriedrichAltmann
Universität für Bodenkultur
Vienna, Austria

FriedemannHesse
Polymun Scientific Immunbiologische Forschungs GmbH
Vienna, Austria

Partners

MarioPezzotti
Università Degli Studi di Verona
Verona, Italy

RainerFischer,JuergenDrossardandStefanSchillberg
Fraunhofer Institute of Molecular Biology and Applied 
Ecology
Aachen, Germany

EugenioBenvenuto
Ente per le Nuove Technologie, l’Energia e l’Ambriente
Rome, Italy

JulianMaandDavidLewis
St George’s Hospital Medical School
London, UK

ChristianVivares
Université Blaise Pascal Clermont-Ferrand II
Aubiere, France

EvaStogerandRainerFischer
Rheinisch-Westfälische Technische Hochschule (RWTH)
Aachen, Germany

HenriSalmon
Institut National de la Recherche Agronomique
Paris, France

PhilipJ.DixandJackieNugent
National University of Ireland
Maynooth, Republic of Ireland

JohnC.GrayandKathrynLilley
University of Cambridge
Cambridge, UK

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IMMUNOTHERAPY ANd TRANSPLANTATION

PaulGarside
University of Glasgow
Glasgow, UK

AnnDepicker
Vlaams Interuniversitair Instituut voor Biotechnologie 
VZW
Zwijnaarde, Belgium

NikosLambrou
Agricultural University of Athens
Athens, Greece

RobEissandHarryThangaraj
Centre for the Management of Intellectual Property in 
Health Research and Development (MIHR)
London, UK

EricvanWijk
Mosaic Systems BV
Prinsenbeek, Netherlands

PaulChristou
Univeritat de Lleida
Lleida, Spain

OscarReif
Sartorius AG
Gottingen, Germany

DavidRobinsion
Ruprecht-Karls-Universität Heidelberg
Heidelberg, Germany

MarcBoutry
Université Catholique de Louvain
Lovain-la-Neuve, Belgium

JurgenDenecke
University of Leeds
Leeds, UK

AlessandroVitale
Consiglio Nazionale Delle Ricerche
Rome, Italy 

RachelChikwambaandEugeniaBarros
Council for Scientific and Industrial Research (CSIR)
Pretoria, South Africa

JohnathanA.Napier
Rothamstead Research Ltd
Harpenden, UK

Jean-MarcNeuhaus
Université de Neuchatel
Neuchatel, Switzerland

RalphBock
Max Planck Institute
Postdam, Germany

TonyKavanagh
Trinity College
Dublin, Republic of Ireland

UdoConrad
Institut für Pflanzengenetik und
 Kulturpflanzenforschung
Gatersleben, Germany

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SAgE

SME-ledantibodyglycol-engineering

ContractNo 
Projecttype

LSHB-CT-2007-037241
SME-SpecificTargeted

 ResearchProject

ECcontribution  e 1843427
Startingdate 1April2007

Duration 36months







Background and objectives:

The  use  of  plant  systems  for  the  production  of 
pharmaceutical  glycoproteins  (including  anti-
bodies) offers an attractive alternative to the cur-
rent state-of-the-art in protein production, given 
their cost efficiency and overall safety. Producing 
large  quantities  of  antibodies  for  use  in  cancer 
diagnostic  and  therapeutic  systems  using  plant 
systems could therefore prove a viable and finan-
cially sound approach.  

However,  the  differences  in  glycan  structures 
added by plants in comparison to those found in 
humans pose significant obstacles and in fact re-
searchers recognise that the plant species used 
as  well  as  the  tissue  and  cell  type  and  the  age 
have a huge impact on the final glycoform of an 
antibody. 

The SAGE project targets an improved plant pro-
duction  platform  for  pharmaceutical  glycopro-
teins. The partners, a high-calibre team of experts 
in  plant-based  production  technology  and  im-
munology,  will  use  four  plant-based  expression 
systems  (including  transgenic  plants,  virus-in-
fected  plants  and  transformed  plant  cell  lines) 
and mammalian cells as a control to generate a 
therapeutic  antibody  that  recognises  the  well-
characterised  carcinoembryonic  antigen  (CEA), 
as  well  as  their  experience  and  know-how  to 
launch protein therapeutics on the market.

four 

The partners, part of an international consortium 
of 
research  organisations,  one  small 
and  medium-sized  enterprise  (SME)  and  two 
companies, will produce an antibody as a panel 
of different glycoforms, which will be tested for 
the following:
• 
•  efficacy  (e.g.  in  Fc-receptor  binding 
assays, tumour cell binding assays and tu-
mour grafting);
•  pharmacokinetic  properties,  such  as 
serum  half  life  and  antibody-dependent 
cellular cytoxicity (ADCC).

stability;

The  project  partners  will  use  the  results  to  de-
velop safer and more active glycoform varieties 
for therapeutic applications. 

Approach and methodology:

By using the plant expression systems and BY-2 
cells to generate the same recombinant antibody, 
the consortium targets the production of H10, a 
human full-length immunoglobulin G (IgG) that 
recognises the CEA. 
SAGE  will  also  conduct  a  comprehensive,  com-
parative  study  to  establish  how  the  structural, 
functional  and  clinical  properties  of  the  anti-
body are influenced by the glycan structures. The 
project  partners  will  compare  the  properties  of 
the H10 antibodies with those of a control H10 
molecule  generated  in  Chinese  Hamster  Ovary 
(CHO) cells.

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IMMUNOTHERAPY ANd TRANSPLANTATION

Expected outcome:

Coordinator

The  use  of  advanced  plant-based  expression 
technologies will help SAGE generate innovative 
therapeutic and diagnostic antibodies. The con-
sortium  will  establish  which  best  plant-based 
expression platform should be used to produce 
therapeutic  antibodies,  as  well  as  identify  the 
glycan  structures  that  give  antibodies  superior 
properties in a clinical setting.

The team will also determine the effects the vari-
ous  plant-derived  glycans  have  on  the  physical 
and functional properties of antibodies. This ac-
tion will support SAGE’s intention to produce im-
proved antibody-based therapeutics with supe-
rior performance, as well as to provide treatment 
for  many  more  people  who  need  it.  Ultimately, 
SAGE  will  be  instrumental  in  improving  health 
care for everyone and in bringing down the costs 
for treatment.

With strong business support, SAGE will convert 
any new knowledge or product developed dur-
ing the duration of the three-year project direct-
ly  into  strategic  advantage. The  project  partner 
Bayer BioScience will utilise IP management sup-
port to conduct IP searches and secure extra IP 
protection, if needed.

SAGE will also transfer the acquired knowledge to 
other scientific groups working in the same field. 
Results and information will be disseminated via 
electronic mail, peer-reviewed articles, abstracts 
and  posters. The  SAGE  consortium  website  will 
also make key findings available to the public.

StefanSchillberg
Fraunhofer-Institut für Molekularbiologie und Ange-
wandte Oekologie IME 
Department of Plant Biotechnology
Forckenbeckstrasse 6 
52074 Aachen, Germany
E-mail: stefan.schillberg@ime.fraunhofer.de

Partners

YuriGleba
ICON Genetics
Halle, Germany

GilbertGorr
greenovation Biotech GmbH
Freiburg, Germany

GerbenvanEldik
Bayer BioScience
Gent, Belgium

DirkBosch,DionFlorack,GerardRouwendal
Plant Research International BV
Wageningen, Netherlands

EvaStogerandRainerFischer
RWTH
Aachen, Germany

HardevSPandhaandDavidLewis
St George’s Hospital Medical School
London, UK

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201

BMC

Bispecificmonoclonalantibodytechnologyconcept

IMMUNOTHERAPY ANd TRANSPLANTATION

ContractNo 
Projecttype

LHSB-CT-200-1818
STREP

ECcontribution  e 240000

Startingdate 1November200

Duration 36months






Background and objectives:

Among  protein-based  drugs,  monoclonal  an-
tibodies  (mAb)  have  a  particular  characteristic, 
acting both as a drug and as a targeted delivery 
system.  MAb  have  recently  shown  great  poten-
tial  in  the  treatment  of  human  cancer  because 
they  are  much  more  specific  than  conventional 
chemotherapy. 

Antibodies are large protein molecules compris-
ing two parts: First, the two variable binding sites 
confer the unique specificity to the molecule, and 
can send a signal of growth inhibition or of pro-
grammed death, called apoptosis. Second, a con-
stant part can trigger the activation of host de-
fence cytotoxic molecules or killer lymphocytes, 
directed against the cancer cells. However, a great 
deal of progress is needed before we can under-
stand the mechanism of binding and the activity 
of antibodies, in order to improve their targeting 
capabilities and their efficiency.

BMC aims to do the following:

•  develop a recombinant bispecific mAb 
with  tetrameric  binding  sites,  directed 
against  two  different  antigens  expressed 
on the same target tumour cell, which will 
add  more  specificity  and  also  double  its 
chances to target tumour cells and to re-
cruit more effector natural killer cells;
•  develop new bispecific or bifunctional 
molecules with the property of cross-link-
ing two different receptors on the surface 

of the cell, an event known to enhance the 
signal transduction pathway of apoptosis;
•  modify  the  carbohydrate  moiety  of 
bispecific antibodies and the tumour tar-
geting  the  complement  regulator  mol-
ecule, properdin, to trigger the activation 
of the complement enzymatic cascade at 
the tumour site; 
•  establish  the  chemical  conjugation 
of  antibodies  of  different  specificities  on 
emulsion nanoparticles. 

Approach and methodology:

The  development  of  novel  approaches  to  opti-
mise the targeting and destruction of tumoural 
cells  is  ensured  in  the  following  ways:  by  engi-
neering  new  constructs  of  recombinant  bispe-
cific  or  bifunctional  antibodies  reacting  with 
two different antigens on the same tumour cells; 
by  developing  new  strategies  for  the  induction 
of  complement-dependant  cytotoxicity  (CDC), 
either  through  modifications  of  the  glycosyla-
tion  of  bispecific  monoclonal  antibodies,  or    by 
genetically  fusing  properdin    to  recombinant 
monoclonal antibodies; and by creating a chemi-
cal conjugation of antibodies of different specifi-
cities on emulsion nanoparticles.

The  consortium  will  evaluate  and  validate  the 
novel  tumour  targeting  strategies  proposed  in 
the  project,  on  tumour  cells  from  patients  with 
CD5+ B-cell lymphoproliferative diseases, as well 
as in an experimental model of SCID mice graft-

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IMMUNOTHERAPY ANd TRANSPLANTATION

ed with CD5+ B-cell lines. The optimisation of the 
benefit/risk ratio in B CD5+ B-cell leukaemias and 
lymphomas will also constitute a key approach. 

Expected outcome:

A doubling of the avidity of bispecific antibodies 
for the BCLL targets tumour cells, in comparison 
with  state-of-the-art  monospecific  monoclonal 
antibodies,  can  be  made  possible  with  through 
the following activities: 

• 
induction  of  apoptosis  in  B-CLL  cells 
previously  unresponsive  to  monomeric 
anti- CD5 mAb;
• 
induction  of  lysis  of  at  least  50%  of 
BCLL cells in the presence of human com-
plement and specific mAbs with carbohy-
drate modification or fused properdin;
• 
induction of C3b opsonisation in 100% 
of BCLL cells in the presence of the same 
reagents;
increase  of  vascular  permeability  in 
• 
subcutaneous  tumour  xenografts  of  hu-
man  BCLL  in  SCID  mice,  after  systemic 
injection  of  mAb  with  a  complement  ac-
tivating property;
• 
inhibit  tumour  growth  in  a  model  of 
subcutaneous  xenografts  of  human  B-
CLL in SCID mice, by systemic injection of 
selected  bispecific  or  bifunctional  mol-
ecules.

Coordinator 

JeanKadouche
Monoclonal Antibodies Therapeutics
Génopole Campus 1
5, rue Henri Desbruères 
91030 Evry cedex, France 
E-mail: j.kadouche@matbiopharma.fr

Partners

Jean-PierreMach
University of Lausanne
Bruno Robert Institute of Biochemistry
Lausanne, Switzerland

ChristianBerthoud
Université de Brest
Brest, France

MartineCerutti
Centre National de la Recherche Scientifique
Montpellier, France

SimonBenita
Hebrew University of Jerusalem
School of Pharmacy
Jerusalem, Israel

JoséeGolay
Ospedali Riuniti di Bergamo
Bergamo, Italy

JurgenBorlak
Fraunhofer – Institut Toxikologie und Experimentelle 
Medizin
Hannover, Germany

CandiceOuinon
ALMA Consulting Group
Lyon, France

PatrickHenno
MABGENE S.A.
Alès, France

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203

 
 
 
 
 
AUTOCURE

Curingautoimmunediseases.Atranslationalapproachtoautoimmunediseasesin
the post-gnomic era using inflammatory arthritis and myositis as prototypes and
learningexamples

IMMUNOTHERAPY ANd TRANSPLANTATION

ContractNo 
Projecttype

LSHB-CT-2006-018661
IntegratedProject

ECcontribution  e 11000000
Startingdate 1March2006
Duration 60months






Website  www.autocure.org

Background and objectives:

The objective of the AutoCure project is to trans-
form  knowledge  obtained  from  molecular  re-
search, particularly within genomics, into a cure 
for  the  increasing  number  of  patients  suffering 
from  inflammatory  rheumatic  diseases.  Rheu-
matoid arthritis (RA) is used as a prototype, since 
this disease offers unique opportunities to define 
and evaluate new therapies. 

Development of the first generation of targeted 
therapies  (anti-TNF  and  anti-IL-1)  in  chronic  in-
flammatory  disease  used  RA  as  the  prototype 
disease,  following  work  by  European  investiga-
tors  included  in  the  current  project.  This  work 
demonstrated the following: 

targeted 

therapies  can  be 

• 
that 
efficient; 
• 
that  a  cure,  while  not  yet  achieved,  is 
within the reach of a strong international 
consortium  covering  translational  re-
search and molecular technology.

Potential  key  molecular  mechanisms  determin-
ing  the  course  of  RA  and  myositis  are  defined 
from  genetic  studies  in  humans,  from  relevant 
animal models and from basic cell and molecu-
lar  biology.  Predictors  of  disease  development 
and therapeutic responses, which could lead to 
future  individualised  therapies,  are  developed 
with  the  help  of  the  consortium’s  unique  large 
patient  group  and  biobanks.  Development  and 
evaluation of new therapies is performed using 

204

New Therapies – Immunotherapy and Transplantation

combinations of novel molecular tools, and pre-
cise definition of disease phenotypes. 

AutoCure’s aims involve producing the following: 
•  an  increased  understanding  of  the 
causes of RA and myositis, enabling better 
prevention; 
•  new potential targets for therapy in ar-
thritis and myositis, which can be further 
tested  in  other  rheumatic  inflammatory 
diseases; 
•  a  prototype  system  for  translational 
research  in  Europe,  enabling  collabora-
tive development of targeted therapies in 
many inflammatory diseases and allowing 
European  SMEs  to  rapidly  develop  ideas 
and patents into targeted therapies in in-
flammatory diseases.

Approach and methodology:

The project targets available knowledge in genet-
ics and genomics, to gain a better understanding 
of rheumatoid arthritis and myositis. This will be 
achieved  through  the  appropriate  use  of  large-
scale genetic investigations to characterise sub-
sets  of  rheumatoid  arthritis  and  myositis,  and 
the  use  of  genetic,  as  well  as  other  molecular 
characteristics,  to  predict  disease  development 
in these different subsets. Subsequently, knowl-
edge  about  molecular  mechanisms  in  different 
genetically  defined  subsets  would  be  used  to 
develop new targeted therapies, suitable for dif-
ferent subsets of these diseases. 

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IMMUNOTHERAPY ANd TRANSPLANTATION

Expected outcome:

The AutoCure consortium expects to deliver new 
and  original  knowledge  concerning  molecular 
pathways  used  for  different  kinds  of  rheumatic 
diseases,  and  also  to  provide  a  platform  for  the 
development  of  new  preventive  measures  and 
therapies.

Main findings:

Findings have been made within the areas of eti-
ology and pathogenesis of arthritis. Using large 
biobanks with information on genetics and envi-
ronmental factors, and immunity, the consortium 
has been able to define new risk factors for RA, 
such  as  smoking  and  other  environmental  risk 
factors,  how  they  interact  with  genes,  and  how 
they  give  rise  to  immune  reactions  that  may 
cause  arthritis.  Immunity  against  citrullinated 
antigens  were  initially  described  by  scientists 
within this consortium, and studies on how such 
immunity can cause arthritis is now very actively 
ongoing within the consortium.

Factors  triggering  the  development  of  myositis 
are investigated in one work package, and new 
findings have been made concerning the molec-
ular  mechanisms  of  development  of  the  symp-
toms of myositis, such as the characteristic weak-
ness  that  causes  disability.  Using  experimental 
models  for  arthritis,  new  modes  of  using  siRNa 
for  inhibiting  cytokine  production  have  been 
used  both  in vitro  and  in vivo.  Many  groups  are 
now collaborating on the greater use of siRNa for 
therapy of arthritis.

The development of therapies continues in many 
of the clinics that are participating in the project. 
In  particular,  studies  on  the  individualisation  of 
therapy are ongoing, where biomarkers and ge-
netic determinants are used to determine the ef-
ficacy, as well as the adverse effects of targeted 
therapies against arthritis and myositis.

• 21 academic institution

• 5 SME’s

• The Karolinska Institute (Co-ordinating institution)

• Prof Lars Klareskog (Coordinating Investigator)

Who are AutoCure 

A major effort is being made apropos the harmo-
nisation of guidelines for collection, and the use 
of large clinical data bases and biobanks in rela-
tion to bioethical rules and regulations in Europe. 
The  guidelines  to  be  developed  will  effectively 
help scientists — both within rheumatology and 
in  other  fields  —  to  work  efficiently  together 
while  adhering  strictly  to  guidelines  and  ethi-
cal  rules.  Collaboration  is  ongoing  between  the 
clinical and experimental academic laboratories, 
as well as the companies contributing to the con-
sortium. Here, in particular, Genmab has been de-
veloping active new therapies with support from 
scientists active in the consortium.

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New Therapies – Immunotherapy and Transplantation

20

IMMUNOTHERAPY ANd TRANSPLANTATION

AUTOCURE

Overview

Major publications

Van  der  Pouw  Kraan,  T.C.,  Wijbrandts,  C.A.,  van 
Baarsen, L.G., Voskuyl, A.E., Rustenburg, F., Baggen, 
J.M., Ibrahim, S.M., Fero, M., Dijkmans, B.A., Tak, P.P., 
Verweij, C.L., ‘Rheumatoid Arthritis subtypes iden-
tified  by  genomic  profiling  of  peripheral  blood 
cells: Assignment of a type I interferon signature 
in  a  subpopulation  of  patients’,  Ann  Rheum  Dis., 
2007, Jan 18;

Michou, L., Croiseau, P., Petit-Teixeira, E., du Mont-
cel, S.T., Lemaire, I., Pierlot, C., Osorio, J., Frigui, W., 
Lasbleiz, S., Quillet, P., Bardin, T., Prum, B., Clerget-
Darpoux,  F.,  Cornelis,  F.,  ‘European  Consortium 
on  Rheumatoid  Arthritis  Families.  Validation  of 
the reshaped shared epitope HLA-DRB1 classifi-
cation  in  rheumatoid  arthritis’,  Arthritis  Res Ther, 
2006, 8(3):R79. 

Working model

Grundtman, C., Salomonsson, S., Dorph, C., Bruton, 
J., Andersson, U., Lundberg, I.E., ‘Immunolocaliza-
tion of interleukin-1 receptors in the sarcolemma 
and  nuclei  of  skeletal  muscle  in  patients  with 
idiopathic  inflammatory  myopathies’,  Arthritis 
Rheum, 2007, Jan 30;56(2):674-687

Hultqvist,  M.,  Olofsson,  P.,  Gelderman,  K.A.,  Hol-
mberg,  J.,  Holmdahl,  R., ‘A  new  arthritis  therapy 
with  oxidative  burst  inducers’,  PLoS  Med,  2006, 
Sep;3(9):e348.

Canete,  J.D.,  Santiago,  B.,  Cantaert,  T.,  Sanmarti, 
R., Palacin, A., Celis, R., Graell, E., Gil-Torregrosa, B., 
Baeten, D., Pablos, J.L., ‘Ectopic lymphoid neogen-
esis in psoriatic arthritis’, Ann Rheum Dis, 2007, Jan 
12;

Wesoly, J., Hu X., Thabet, M.M., Chang, M., Uh, H., 
Allaart, C.F., Toes, R.E., Houwing-Duistermaat, J.J., 
Begovich, A.B., Huizinga, T.W., ‘The 620W allele is 
the PTPN22 genetic variant conferring suscepti-
bility to RA in a Dutch population’, Rheumatology, 
Oxford, 2006, Nov 29.

Bendtzen, K., Geborek, P., Svenson, M., Larsson, L., 
Kapetanovic, M.C., Saxne, T., ‘Individualized moni-
toring of drug bioavailability and immunogenic-
ity in rheumatoid arthritis patients treated with 
the tumor necrosis factor alpha inhibitor inflixi-
mab’, Arthritis Rheum, 2006, Dec; 54(12):3782-9.

Michou,  L.,  Lasbleiz,  S.,  Rat,  A.C.,  Migliorini,  P., 
Balsa, A., Westhovens, R., Barrera, P., Alves, H., Pier-
lot, C., Glikmans, E., Garnier, S., Dausset, J., Vaz, C., 
Fernandes,  M.,  Petit-Teixeira,  E.,  Lemaire,  I.,  Pas-
cual-Salcedo,  D.,  Bombardieri,  S.,  Dequeker,  J., 
Radstake, T.R., Van Riel, P., van de Putte, L., Lopes-
Vaz, A., Prum, B., Bardin, T., Dieude, P., Cornelis, F., 
‘European  Consortium  on  Rheumatoid  Arthritis 
Families.  Linkage  proof  for  PTPN22,  a  rheuma-
toid  arthritis  susceptibility  gene  and  a  human 
autoimmunity gene’, Proc Natl Acad Sci USA, 2007, 
Jan 30;104(5):1649-54.

206

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IMMUNOTHERAPY ANd TRANSPLANTATION

Coordinator
LarsKlareskog
Karolinska Institutet
Department of Medicine
Rheumatology Unit
Stockholm, Sweden
E-mail lars.klareskog@ki.se 
or Susanne.karlfeldt@karolinska.se 

Partners

TomHuizinga
Leiden University Medical Centre
Leiden, Netherlands

GerdBurmesterandAndreasRadbruch
Charité Universitätsmedizin Berlin
Berlin, Germany

JosefSmolen
Medical University of Vienna
Vienna, Austria

BarryBresnihan
St Vincent’s University Hospital
Dublin, Ireland

PaulPeterTakandDominiqueBaeten
Univesrity of Amsterdam
Amsterdam, Netherlands

AndrewCope
Imperial College
London, UK

WimvandenBergandWalthervanVenrooij
University Medical Centre
Nijmegen, Netherlands

ChristianJorgensen
Institut de la santé et de la recherche medicale
Paris, France

RenateGay
University of Zurich
Centre of Experimental Rheumatology 
and WHO Collaborating Centre
Zurich, Switzerland

ToreSaxneandRikardHolmdahl
Lund University
Lund, Sweden

SolbrittRantapää-Dahlqvist
Umeå University 
Umeå, Sweden

JiriVencovsky
Institute of Rheumatology
Prague, Czech Republic

JaneWorthington
University of Manchester
Manchester, UK

JanvandeWinkel
GENMAB A/S
Copenhagen, Denmark

ChristopherBuckley
University of Birmingham
Birmingham, UK

FrançoisCornelis
Univeristé d’Evry
Evry, France

WlodzimierzMaslinski
Institute of Rheumatology
Warsaw, Poland

DimitriosBoumpas
University of Crete
Heraklion, Crete, Greece

GerdBurmester
EULAR Genomics Study Group
Zurich, Switzerland

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IMMUNOTHERAPY ANd TRANSPLANTATION

AUTOCURE

CorVerweij
VU University Medical Centre
Amsterdam, Netherlands

PeterOlofsson
Biovitrum
Gothenburg, Sweden

KarineMignon-Godefroy
BMD
Marne-la-Vallée, France

MargrietVervoordeldonk
Arthrogen B.V.
Amsterdam, Netherlands

208

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IMMUNOTHERAPY ANd TRANSPLANTATION
INNOCHEM

Innovative chemokine-based therapeutic strategies for autoimmunity and
chronicinflammation

Innochem






ContractNo 
Projecttype

LSHB-CT-200-18167
IntegratedProject

ECcontribution  e 11730696

Startingdate 1November200

Duration 60months

Website  www.altaweb.eu/innochem

Background and objectives:

The general objective of INNOCHEM is to develop 
innovative chemokine-based therapeutic strate-
gies for autoimmunity and chronic inflammation. 
The project is based on the scientific excellence 
of  the  applicants,  which  have  made  major  rec-
ognised contributions to the field since the very 
beginning  of  chemokine  discovery,  and  on  the 
construction  of  shared  technological  platforms. 
This includes the following: 
•  proteomics; 
• 
transcriptional profiling for the outline 
of the ‘chemokinome’ in pathophysiologi-
cal  conditions  and  identification  of  new 
antagonists; 
•  molecular modelling of agonist/antag-
onist receptor or agonist/inhibitor interac-
tion, for pharmacology and drug design; 
•  gene modified mice for target valida-
tion in autoimmune disorders. 

Genetic,  structural,  biological,  and 
immun-
opathological  studies  will  provide  a  framework 
for  the  development  of  innovative  chemokine-
based therapeutic strategies. The therapeutic ap-
proaches  to  be  investigated  are  innovative  and 
not  limited  to  conventional  antagonists.  These 
include  decoy  receptors,  agonist  binders,  and 
non-competitive allosteric inhibitors. In addition 
to  academic  groups,  therapy-oriented  research 
includes 3 biotech SMEs, 1 medium- and 2 large-
sized pharmaceutical companies. The companies 
involved  are  developing  complementary  non-

overlapping  approaches  to  target  the  chemok-
ine system with recombinant and low molecular 
weight molecules. 

INNOCHEM  is  expected  to  conduct  a ‘proof-of-
principle’  clinical  study  in  volunteers. The  ambi-
tion  of  this  project  is  to  re-establish  European 
leadership  in  basic  and  applied  chemokine  re-
search,  by  integrating  academic  and  industrial 
cutting-edge groups, to develop innovative ther-
apeutic  strategies  against  autoimmunity  and 
chronic inflammatory disorders.

Approach and methodology:

The  approach  of  INNOCHEM  is  based  on ‘plat-
forms’,  a ‘pipeline’  and  the ‘integration  of  excel-
lence’. The hard core ‘platforms’ on which INNO-
CHEM is based, include the following:

•  proteomics  and  whole  genome  tran-
scriptional  profiling  for  the  definition  of 
the  chemokinome  in  pathophysiological 
conditions,  and  for  the  identification  of 
new modulators;
•  gene modified mice, including double 
knock  outs  (KO)  and  KO  mice  in  autoim-
mune  backgrounds  for  pathophysiology, 
target  identification  and  validation,  and 
preclinical evaluation;
•  molecular  modelling  for  agonist/an-
tagonist-receptor  interaction  or  agonist-
inhibitor  interaction  and  pharmacology 
for the design and optimisation of innova-
tive strategies.

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209

 
 
 
 
 
 
 
 
 
 
IMMUNOTHERAPY ANd TRANSPLANTATION

INNOCHEM

‘pipeline’  characteristics  are  presented 

The 
below:

Expected outcome:

•  Development  of  original,  anti-chem-
okine  strategies  including  receptor  an-
tagonists, agonist blockers, competitors of 
agonist-GAG interaction, decoy receptors, 
and non-competitive allosteric inhibitors. 
These strategies are original inasmuch as 
they extend beyond (but include) conven-
tional antagonists.
•  Pathophysiology  of  the  chemokine 
system  in  preclinical  and  clinical  condi-
tions, as a basis for rational, targeted inter-
vention.
•  Preclinical,  multistep  evaluation,  in-
cluding target validation in gene modified 
mice and by genetic analysis in humans
•  Proof-of-principle clinical studies.

 The entire pipeline has been activated from the 
very  beginning  of  the  study  (Fig  1). ‘Integration 
of  excellence’  represents  a  major  characteristic 
of  the  INNOCHEM  approach.  Not  only  does  IN-
NOCHEM have the ambition of collecting and at-
tracting major European groups in the field, but 
its components also have a proven strong track 
record of bilateral interactions. In the context of 
INNOCHEM, a quantum leap of integrating Euro-
pean efforts in the field will be possible, on the 
solid basis of a tradition of collaboration.

The goal of INNOCHEM is to develop innovative 
chemokine inhibitors as a novel therapeutic ap-
proach  for  the  treatment  of  autoimmune  and 
chronic inflammatory diseases up to one ‘proof-
of-concept’  study  in  humans. The  development 
of innovative therapeutic strategies will be based 
on a definition of pathophysiological significance 
and target validation (Fig 2). The therapeutic ap-
proaches to be investigated are innovative, and 
not  limited  to  conventional  antagonists.  These 
will include decoy receptors, agonist binders, and 
non-competitive allosteric inhibitors. 

A  unique  non-competitive  allosteric  inhibitor, 
Reparixin, which has completed phase I and has 
been  awarded  orphan  drug  status  by  FDA  and 
EMEA, is available for proof-of-concept studies in 
humans.

The results obtained in this project will open new 
perspectives in therapeutic intervention against 
autoimmunity  and  chronic  inflammation,  with 
social  impacts  measurable  as  improved  health 
and reduced health societal costs, (including di-
rect  and  indirect  costs  for  patient  assistance,  as 
well as loss of manpower), emerge.

Figure 1. – INNOCHEM pipeline.

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IMMUNOTHERAPY ANd TRANSPLANTATION

Main findings:

During  the  first  year  of  activity,  the  INNOCHEM 
consortium  focused  its  efforts  along  the  lines 
originally  identified,  albeit  with  some  adjust-
ments.  In  general,  the  lines  of  activity  encom-
passed diverse fields, ranging from the basic bi-
ology  of  the  chemokine  system  to  translational 
efforts in the clinical phase. Potential technologi-
cal  approaches  have  included  the  use  of  whole 
genome  transcriptional  profiling,  the  use  of 
gene-modified mice, and original reagents gen-
erated by the group. A major thrust of the INNO-
CHEM  efforts  has  been  devoted  to  strengthen-
ing integration and collaboration. As an example, 
one could emphasise the strengthening of Euro-
pean leadership in the field of silent receptors for 
chemokines which can act as decoys. In terms of 
translational efforts, a major result obtained dur-
ing the first year is the identification of a chem-
okine  (CXCL8)  as  a  surrogate  endpoint  for  the 
activity in patients in inflammatory prostatitis of 
a compound currently being developed by one 
of the participating SMEs.

Major publications 

Pello, O.M., Moreno-Ortiz, M.D., Rodriguez-Frade, 
J.M., Martinez-Munoz, L., Lucas, D., Gomez, L., Lu-
cas, P., Samper, E., Aracil, M., Martinez, A., Bernad, 
A.,  Mellado,  M.,  ‘SOCS  upregulation  mobilises 
autologous stem cells through CXCR4 blockade’, 
Blood, 2006, 108:3928-3937.

Rosenkilde,  M.M.,  David,  R.,  Oerlecke,  I.,  ned-
Jensen,  T.,  Geumann,  U.,  Beck-Sickinger,  A.  G., 
Schwartz,  T.W.,  ‘Conformational  constraining  of 
inactive  and  active  states  of  a  7TM  receptor  by 
metal-ion  site  engineering  in  the  extracellular 
end  of  transmembrane  segment  V  (TM-V)’,  Mol 
Pharmacol, 2006, 70:1892-1901.

Penna,  G.,  Mondaini,  N.,  Amuchastegui,  S.,  Degli 
Innocenti, S., Carini, M., Giubilei, G., Fibbi, B., Colli, 
E.,  Maggi,  M.,  Adorini,  L.,  ‘Seminal  plasma  cy-

Figure 2. - Outline of INNOCHEM targeting chemokine/re-
ceptor interactions, emphasising the implementation of 
therapeutic strategies.

tokines  and  chemokines  in  prostate  inflamma-
tion:  Interleukin  8  as  a  predictive  biomarker  in 
chronic prostatitis/chronic pelvic pain syndrome 
and benign prostatic hyperplasia’, Eur Urol, 2007; 
Feb;51(2):524-33.

Schmid,  H.,  Boucherot,  A.,  Yasuda,  Y.,  Henger,  A., 
Brunner, B., Eichinger, F., Nitsche, A., Kiss, E., Bleich, 
M., Grone, H. J., Nelson, P. J., Schlondorff, D., Cohen, 
C.  D.,  Kretzler,  M., ‘Modular  activation  of  Nuclear 
Factor-ΔB  transcriptional  programs  in  human 
diabetic  nephropathy’,  Diabetes,  2006,  55:2993-
3003.

Proost, P., Struyf, S., Loos, T., Gouwy, M., Schutyser, 
E., Conings, R., Ronsse, I., Parmentier, M., Grillet, B., 
Opdenakker, G., Balzarini, J., Van Damme, J., ‘Coex-
pression and interaction of CXCL10 and CD26 in 
mesenchymal cells by synergising inflammatory 
cytokines: CXCL8 and CXCL10 are discriminative 
markers for autoimmune arthropathies’, Arthritis 
Res Ther, 2006, 8:R107.

Springael,  J. Y.,  Le  Minh,  P.  N.,  Urizar,  E.,  Costagli-
ola, S., Vassart, G., Parmentier, M., ‘Allosteric mod-
ulation  of  binding  properties  between  units  of 
chemokine receptor homo- and hetero-oligom-
ers’, Mol Pharmacol, 2006, 69:1652-1661.

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IMMUNOTHERAPY ANd TRANSPLANTATION

INNOCHEM

Sironi, M., Martinez, F.O., D’Ambrosio, D., Gattorno, 
M., Polentarutti, N., Locati, M., Gregorio, A., Iellem, 
A.,  Cassatella,  M.A.,  Van  Damme,  J.,  Sozzani,  S., 
Martini, A., Sinigaglia, F., Vecchi, A., Mantovani, A., 
‘Differential regulation of chemokine production 
by  FcΔ  receptor  engagement  in  human  mono-
cytes: association of CCL1 with a distinct form of 
M2 monocyte activation (M2b, type 2)’,  J Leukoc 
Biol, 2006, 80:342-349.

Coordinator 

AlbertoMantovani
Humànitas Mirasole S.p.A
Via Manzoni 56
20089 Rozzano-Milano, Italy
E-mail: alberto.mantovani@humanitas.it

Scientific coordinator 

MariagraziaUguccioni
Istituto di Ricerca in Biomedicina
Via Vela 6 
6500 Bellinzona, Switzerland
E-mail: mariagrazia.uguccioni@irb.unisi.ch

Partners 

ChristopheCombadiere
Université Pierre et Marie Curie-Paris
INSERM U543
Paris, France

DominiqueEmilie
INSERM 
Paris, France

AndrzejGlabinski
Uniwersytet Medyczny w Łodzi, Department 
of Neurology
Lodz, Poland

MartinLipp
Max-Delbrueck-Centrum für Moleckulare Medizin
Berlin, Germany

CarlosMartinez
Centro Nacional de Biotecnologia, CSIC
Madrid, Spain

BernhardMoser
Universität Bern, Institute of Cell Biology
Bern, Switzerland

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IMMUNOTHERAPY ANd TRANSPLANTATION

PeterJ.Nelson
Ludwig-Maximilians-University Münich
Münich, Germany

AntalRot
Novartis Institutes for Biomedical Research
Vienna, Austria

AmandaProudfoot
Serono International SA
Plan les Outas, Switzerland

AldoTagliabue
Alta S.r.l.
Siena, Italy

MarcParmentier
Université Libre de Bruxelles, IRIBHM
Brussels, Belgium

CostantinoPitzalis
King’s College London
London, UK

SergioRomagnani
Università degli Studi di Firenze, “MCIDNENT”
Florence, Italy

MetteM.Rosenkilde
University of Copenhagen
The Panum Institute
Department of Pharmacology
Copenhagen, Denmark

JozefvanDamme
Katholieke Universiteit
Leuven, Belgium

TimothyWilliams
Imperial College of Science, Technology and Medicine
London, UK

AmnonPeled
BioKine Therapeutics Ltd.
Rehovot, Israel

LucianoAdorini
BioXell
Milan, Italy

MarcelloAllegretti
Dompe pha.r.ma Spa
L’Aquila, Italy

JanW.Vrijbloed
Polyphor Ltd.- PEMB Unit
Allschwill, Switzerland

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213

CELLAId 

Europeansymposiafortheevaluationofpotentialsandperspectivesofcurative
celltherapiesforautoimmunediseases

IMMUNOTHERAPY ANd TRANSPLANTATION






ContractNo 
Projecttype

LSHB-CT-2004-00094
SpecificSupportAction

ECcontribution  e 198800

Startingdate 1January200
Duration 30months

Website  www.cellaid-eu.org

Background and objectives:

Autoimmune  diseases,  such  as  inflammatory 
rheumatic  diseases,  are  currently  impossible  to 
cure with either conventional therapies or mod-
ern  biologics  therapies.  Existing  therapies  are 
immune-suppressive,  systemic  and/or  accom-
panied  by  serious  side  effects,  which  drastically 
reduce quality of life and life expectancy. The so-
cioeconomic impact of inflammatory rheumatic 
diseases  is  approximately  Δ15  billion  per  year  in 
Germany alone, with a trend for costs to increase 
rapidly due to the market prices of emerging bio-
logics.

The  CELLAID  project  will  establish  a  European 
consortium  to  organise  three  symposia  on  im-
munocyte-based  therapies 
for  autoimmune 
diseases.  The  new  treatment  concepts  should 
target pathomechanisms, especially the involved 
immune  cells  and  messenger  biomolecules  in 
the  course  of  the  disease.  CELLAID  also  aims  at 
providing  a  communication  platform  for  Euro-
pean  experts  to  evaluate  the  existing  potential, 
and develop integrated pan-European strategies. 
Joint European efforts are required to strengthen 
the  European  lead  in  respective  technologies, 
and also to combine experience in pan-Europe-
an clinical trials.

The European Research Area can maintain excel-
lence, and scientific, technological and economic 
competitiveness  with  innovative  therapies  for 
through  networking. 
autoimmune  diseases 

Such  networking  of  European  research  institu-
tions and clinics will enhance research expertise, 
the standardisation of clinical interventions and 
quality assessment, e.g. immune monitoring. Pa-
tient cohorts will grow, even for rare autoimmune 
diseases. The translation of concepts into clinics 
will involve extensive industry cooperation: joint 
efforts  will  boost  the  competitiveness  of  small, 
medium  and  large  biotech  and  pharmaceutical 
companies in Europe. 

Approach and methodology:

The  first  two  CELLAID  symposia  were  held  in 
Berlin in June 2005, and in Brussels in April 2006. 
Cellular  concepts  of  immune  therapies  for  au-
toimmune  diseases  and  European  perspectives 
towards  immunocyte-based  therapies  were  ad-
dressed  by  an  international  selection  of  expert 
speakers,  chairmen  and  participants.  Results 
(programmes  and  summaries)  of  the  symposia 
are published on the website of the project, www.
cellaid-eu.org. The third symposium is scheduled 
to take place in Florence, Italy, on 20-22 February 
2007.

Expected outcome:

Currently,  no  cure  is  available  for  autoimmune 
diseases, which require a life-long immune sup-
pressive  treatment  with  a  high  risk  of  adverse 
events,  and  lack  a  regenerative  perspective.  Im-
munocyte-based  therapies  —  supported  by  re-
cent  advances  made  in  genomics,  immunology 

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IMMUNOTHERAPY ANd TRANSPLANTATION

and cytometry — aim for an autoimmunity cure 
and a reset of tolerance, which is a prerequisite for 
regenerative therapies. They will have high trans-
lational potential for other immune diseases. 

Main findings: 

While  research  highlights  of  the  two  symposia 
were numerous, only a selection is mentioned in 
this  report.  Detailed  summaries  are  available  at 
www.cellaid-eu.org.  The  function  and  dysfunc-
tion of regulatory T cells were the areas of focus 
in a number of presentations. Cytokines such as 
IL-6 and its transsignaling mechanisms were pre-
sented, representing potential new future thera-
peutic  targets.  Many  other  relevant  cytokines 
and their functions were discussed, highlighting 
the  role  of  special  cytokine  networks  that  per-
petuate chronic inflammation, particularly in au-
toimmunity. B cells and their roles as plasma and 
memory cells were intensively discussed in both 

CellAid 2005 - Andreas Radbruch (Coordinator)

symposia.  Currently,  B  cell  (e.g.  CD  20)-targeted 
biologics do achieve the most convincing thera-
peutic  effects.  However,  it  remains  to  be  seen 
whether  remission  or  a  cure  can  be  achieved 
without long-term side effects. 

With regard to genomics in multi-factorial diseas-
es, such as rheumatoid arthritis, the identification 
of genes other than the major histocompatibility 
complex that regulate autoimmune diseases was 
discussed.  One  regulator  of  arthritis  severity  in 
rats was identified and then presented at the first 
CELLAID  symposium.  In  experimental  models,  a 
single gene such as AIRE has provided important 
insight into the regulation of autoimmunity. Dis-
cussions at the two CELLAID symposia highlight-
ed that the power and impact of genetic analy-
sis  is  undisputed,  and  that  the  translation  from 
animal models to genetic analysis for diagnosis, 

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New Therapies – Immunotherapy and Transplantation

21

IMMUNOTHERAPY ANd TRANSPLANTATION

CELLAId

prediction and therapy of autoimmune diseases 
patients is in progress. 

One  of  the  experimental  therapies  gaining  ap-
proval  is  autologous  stem  cell  transplantation, 
performed  for  many  autoimmune  diseases.  In 
Europe,  they  are  registered  through  the  EULAR 
and  EBMT  working  parties. They  do  in  some  in-
stances (e.g. Systemic Lupus Erythematosis) have 
the potential to induce full remission, even in pa-
tients with a long disease history. As discussed in 
the presentations at both meetings, this is seem-
ingly based on the combination of a drastic de-
pletion of pathogenic memory and effector cells, 
and the development of a new and tolerant im-
mune  system.  So  far,  however,  autologous  stem 
cell transplantation is only applicable for stand-
ard  treatment  of  refractory  patients,  as  the  side 
effects can be hazardous.

‘targets’  and 

Finally, mesenchymal stem cells were introduced 
they  have  been  applied 
as 
successfully 
in  experimental  animal  models, 
where  they  show  they  can  provide  strong 
immunosuppressive effects. Results from the first 
European trials in human were presented at the 
CELLAID symposia.

Coordinator

AndreasRadbruch
Deutsches Rheuma-Forschungszentrum
Charitéplatz 1
10117 Berlin, Germany
E-mail: radbruch@drfz.de 

Scientific coordinator

JuttaSteinkötter
Competence network rheumatology
Luisenstr. 41
10117 Berlin, Germany
E-mail: jutta.steinkoetter@dgrh.de

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IMMUNOTHERAPY ANd TRANSPLANTATION
STEMdIAgNOSTICS

Thedevelopmentofnewdiagnostictests,newtoolsandnon-invasivemethods
for the prevention, early diagnosis and monitoring for haematopoietic stem
celltransplantation

ContractNo 
Projecttype

LSHB-CT-2007-037703
SME-SpecificTargetedResearchProject

ECcontribution  e 200000
Startingdate 1June2007

Duration 36months






Background and objectives:

Over 7 000 allogeneic haematopoietic stem cell 
transplants  (HSCT)  are  carried  out  each  year  in 
Europe alone, as a treatment for leukaemia and 
lymphoma.  Techniques  and  cure  rates  are  im-
proving, but the overall survival rate remains be-
tween 40% and 60%. 
STEMDIAGNOSTICS will develop new proteomic, 
biological and genomic tests and tools for early 
diagnosis and monitoring of patient response to 
novel therapeutics for the most severe complica-
tion of HSCT - graft versus host disease (GvHD). 
It  will  bring  to  the  clinic  a  new  generation  of 
diagnostics that will significantly improve HSCT 
therapy and patient outcome.

The consortium unites 5 European SMEs with ex-
pertise  and  markets  in  genomic  and  proteomic 
testing,  diagnostic  assay  development  and  bio-
chips,  with  clinical  partners  selected  for  their 
world  leading  research  in  HSCT  and  access  to 
clinical samples and patient groups. The project 
will  focus  on  the  role  of  relevant  genes  and  bi-
omarkers  associated  with  acute  and  chronic 
GvHD,  using  retrospective  samples  from  estab-
lished biobanks and prospective clinical trials to: 
identify novel bio and genomic mark-

• 
ers for diagnostics;
•  develop  novel  diagnostic  tools  using 
genomics,  proteomics,  in  vitro  bioassays 
and biochips; 
• 
models and on clinical samples;

test  the  new  diagnostics  in  animal 

•  exploit  the  new  tools  for  commercial 
use.

STEMDIAGNOSTICS will develop diagnostic tests 
using  single  nucleotide  polymorphism  (SNP) 
analyses (IMGM), based on results from previous 
EC-funded  research  (Eurobank,  Transeurope). 
It  will  use  proteomics  via  mass  spectrometry 
evaluation/development  of  diagnostic  patterns 
(Mosaiques),  ELISA  kits  (Apotech)  and  protein 
biochip prototypes (Orla) for the development of 
fast, high throughput technologies. The consorti-
um will also develop novel reagents for monitor-
ing  graft  versus  leukaemia,  GvHD  and  targeted 
therapy (Multimune; Nascacell). Finally, compara-
tive studies in an autoimmune disease model of 
inflammation — rheumatoid arthritis — will be 
made.

Adult  stem  cells  are  starting  to  be  used  for  the 
therapy  of  diverse  disorders  including  autoim-
mune  disease  and  cancer.  The  potential  differ-
entiation  of  such  stem  cells  into  various  types 
of adult tissue is also a new and exciting area of 
research.  It  will  create  opportunities  for  the  in-
novation  of  a  new  generation  of  medical  diag-
nostic  tools,  tests  and  therapies;  for  improving 
healthcare  services  and  patient  outcomes;  and 
for reducing expenditure on healthcare systems. 
It  offers  Europe’s  biotech  SMEs  the  opportunity 
to  strengthen  their  competitiveness  and  suc-
cessfully help meet the growing demands of the 
healthcare sector.
Stem  cell  research  is  essential  due  to  the  lack, 

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217

 
 
 
 
 
 
IMMUNOTHERAPY ANd TRANSPLANTATION

STEMdIAgNOSTICS

over  the  past  30  years,  of  any  general  cure  for 
various illnesses, such as cancer, heart disease or 
autoimmune disorders, among others. 

Haematopoietic stem cell transplantation (HSCT), 
however, is one area of stem cell research where 
major advances in the cure of haematological dis-
orders,  like  leukaemia  and  lymphoma,  inherited 
immune  disorders,  and  aplastic  anaemia,  have 
been  made. Transplantation  of  stem  cells  in  this 
context, where immature cells develop into white 
and red blood cells and platelets (i.e. haematopoi-
esis)  from  related  or  unrelated  donors  leads  to 
immune  reconstitution  and  can  be  a  life  saving 
therapy. Currently over 7 000 transplants are car-
ried out each year in Europe. Despite this success 
the overall survival rate after HSCT is poor. 

A  40-60%  survival  rate  is  the  norm  for  such 
transplants, which involve the use of bone mar-
row,  peripheral  blood  stem  cells  and  umbilical 
cord  blood  as  the  stem  cell  source. The  cure  of 
patients is limited by clinical complications that 
arise post-transplant. These are largely due to a 
lack  of  understanding  of  acceptance  and  rejec-
tion  mechanisms  and  genetic  differences  that 
exist  between  a  given  patient  and  donor  and 
inaccurate/lack of diagnostic tests for early com-
plications. 

The application of HSCT is also hampered by the 
lack of suitably matched donors. Only 25-30% of 
patients  will  find  a  HLA  matched  sibling  donor 
and, therefore, more matched unrelated donors 
within  International  Bone  Marrow  Transplant 
Registries  are  needed. There  is  therefore  an  ur-
gent need to improve patient-donor matching at 
both the biological response and genomic level, 
which will increase the pool of both suitable and 
more  appropriate  donors  and  reduce  the  most 
severe  complication  of  acute  and  chronic  graft 
versus host disease (GvHD).

It  is  essential  to  predict  the  development,  the 
severity and treatment outcome of GvHD, which 

is  an  important  predictor  of  transplant  survival. 
The strongest predictor of GvHD is the degree of 
mismatching between patient and donor; how-
ever, there is a significant incidence of acute and 
chronic GVHD even in patients undergoing HLA 
identical sibling transplants, thus clearly indicat-
ing a role of genetic risk factors other than major 
HLA differences There are also no reliable predic-
tive  or  diagnostic  indicators  to  date  for  either 
acute  or  chronic  GvHD  and  no  reliable  markers 
that distinguish GvHD from viral or other inflam-
matory manifestations.

 The most reliable clinical predictors of GvHD are 
the donor recipient sex-mismatch (female donor 
to  male  recipient)  but  no  other  clinical  factors 
can predict acute GvHD. Research therefore has 
focused on the biology of GvHD and the involve-
ment of cytokines in the “cytokine storm”). Serum 
levels of cytokines, mRNA expression of candidate 
cytokines in peripheral blood or target tissue, or 
as in the case of several of the current partners, 
a  study  of  non  HLA  genetic  polymorphisms  for 
candidate  cytokines  using  pretransplant  recipi-
ent  and  donor  DNA  have  been  analysed.  How-
ever,  none  have  as  yet  been  tested  effectively 
for  commercial  use  as  novel  diagnostic  tests). 
These studies will form the basis of further devel-
opment  of  novel  diagnostics  based  on  the  fact 
that  early  detection  of  GvHD  and  its  severity  is 
urgently needed and that its progression needs 
to be monitored during the course of HSCT.

Approach and methodology:

The  project  will  focus  on  the  role  of  relevant 
genes and biomarkers associated with acute and 
chronic GvHD, using retrospective samples from 
established biobanks and prospective clinical tri-
als to: 

identify novel bio and genomic mark-

• 
ers for diagnostics;
•  develop  novel  diagnostic  tools  using 
genomics  proteomics,  in  vitro  bioassays 
and biochips;

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IMMUNOTHERAPY ANd TRANSPLANTATION

test  the  new  diagnostics  in  animal 

• 
models and on clinical samples;
•  exploit  the  new  tools  for  commercial 
use;
•  undertake  comparative  studies  in  an 
autoimmune disease model of inflamma-
tion; rheumatoid arthritis.

The SMEs’ specific aims include: 

•  development  of  new  tools  for  novel 
diagnostics, novel drug targets and thera-
peutics for use in both the transplant and 
autoimmune settings;
•  access  to  important  clinical  and  bio-
logical samples, as well as data for use in 
accurate  assessment  of  results  and  con-
firmatory studies for the development of 
novel diagnostics;
testing  and  evaluation  of  new  diag-
• 
nostics on independent cohorts and cor-
relation of data across HSCT centres;
testing  of  new  prototypes  against 
• 
current  assays  via  collaboration  between 
SMEs;
•  use  of  new  target  molecules  in  the 
monitoring  of  response  to  therapy  and 
during  the  post  transplant  period  to  as-
sess acute and chronic GvHD.

Expected outcome: 

STEMDIAGNOSTICS  will  identify  new  diagnos-
tics in the form of novel proteins associated with 
graft  versus  host  disease  (GvHD)  compared  to 
viral  disease.  The  consortium  will  also  develop 
early diagnostic tools for GvHD and rheumatoid 
arthritis using gene profiling. The fast throughput 
development  of  novel  monoclonal  antibodies 
and ELISA kits for research, diagnostics, and po-
tentially therapeutic use emerge as well. STEMDI-
AGNOSTICS will develop novel peptides for use 
in monitoring GvHD and graft versus leukaemia 
effects in transplant patients, and it will identify 
new single nucleotide polymorphisms (SNPs) for 
analysis in prognostic/diagnostic indices.

Coordinator

AnneDickinson
University of Newcastle upon Tyne
School of Clinical and Laboratory Sciences
The Medical School
Newcastle upon Tyne, UK
E-mail: a.m.dickinson@ncl.ac.uk

Partners

ErnstHoller
Klinikum der Universität Regensburg
Dept. Hamatology und Internistiche Onkologie
Regensburg, Germany

HaraldMischak
Mosaiques Diagnostics GmbH
Hannover, Germany

GabrieleMulthoff
Multimmune GmbH
Munich, Germany

RalphOehlmann
IMGM Laboratories
Martinstried, Germany

LarsFrench
Zurich University Hospital
Department of Dermatology
Zürich, Switzerland

OlivierDonzé
Apotech Corporation (Headquarters)
Epalinges, Switzerland

Hans-JochemKolb
Clinical Cooperation Group Hematopoietic Cell
Transplantation, Institute of Molecular Immunology
Forschungszentrum fuer Umwelt und Gesundheit
Munich, Germany

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219

 
 
 
 
 
 
 
 
STEMdIAgNOSTICS

IMMUNOTHERAPY ANd TRANSPLANTATION

DaleAthey
Orla Protein Technologies Ltd
Nanotechnology Centre
University of Newcastle upon Tyne,
Newcastle, UK

GerardSocie
Association de Recherche sur la Greffe de CSP
Aplosies et HPN
Paris, France

HildegardGreinix
University Hospital of Vienna
Bone Marrow Transplantation Unit
Vienna, Austria

IlonaHromadníková
Charles University
3rd Medical Faculty
Prague, Czech Republic

AmandaMcMurray
CENAMPS
The Fabriam Centre
Atmel Way
Newcastle upon Tyne, UK

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IMMUNOTHERAPY ANd TRANSPLANTATION
RISET

Reprogrammingtheimmunesystemfortheestablishmentoftolerance

ContractNo 
Projecttype

LSHB-CT-200-12090
IntegratedProject

ECcontribution  e 10000000
Startingdate 1March200
Duration 60months






Website  www.risetfp6.org

Background and objectives:

RISET  aims  at  inducing  tolerance,  which  is  de-
fined as the permanent acceptance of the trans-
plant in the absence of continuous immunosup-
pression. It is based on the translation of recent 
advances in post-genomic immunology that are 
focused on the development of new biotechnol-
ogy  products  to  promote  long-term  transplant 
acceptance in preclinical models. 

In  order  to  achieve  this  objective,  the  RISET 
programme  anticipates  developing  diagnostic 
tests to identify transplanted patients for whom 
immunosuppressive  treatment  could  be  safely 
minimised  or  withdrawn.  Related  ethical  and 
societal questions will be specifically addressed, 
in  addition  to  communication  with  patient 
organisations  and  regulatory  bodies.  In  parallel, 
relevant  models  of  tolerance  will  be  used  to 
identify new genes, molecules or cell types that 
will  form  the  basis  for  novel  diagnostic  and 
therapeutic approaches.

The use of knowledge will be facilitated by the di-
rect involvement of SMEs in the consortium, the 
building of an industry platform, and the yearly 
monitoring  of  external  reviewers  with  both  sci-
entific and industrial backgrounds in the field.

Expected outcome:

The  ultimate  goal  of  this  project  is  to  develop 
safe and efficient diagnostic tools, and therapies 

Université Libre de Bruxelles- IMI. Gosselies. Belgique.

to  induce  long-term  tolerance  in  transplanted 
patients, in the absence of any immunosuppres-
sion.  This  ambitious  objective  will  be  achieved 
through  a  multistep  approach  involving  inter-
mediate milestones: 

• 
the  development  of  biological  tests 
predictive of transplant tolerance or ‘near-
tolerance’;
• 
the clinical and biological assessment 
of  the  outcome  of  patients  enrolled  in 
clinical investigations, targeting the com-
plete withdrawal of immunosuppression;
• 
the establishment of ethical guidelines 
for tolerance induction protocols and edu-
cational programmes on tolerance induc-
tion for patients and their families;
the establishment of educational pro-
• 
grammes on tolerance induction for phy-
sicians, scientists and nurses;
the  identification  of  new  molecular 
• 
targets for tolerance induction in preclini-
cal models.

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221

 
 
 
 
 
 
 
 
RISET

IMMUNOTHERAPY ANd TRANSPLANTATION

ary 2006, 2 more patients were transplanted, and 
relevant data will be provided in the next report. 
Identification  and  recruitment  of  additional  pa-
tients  to  be  involved  over  the  second  period  is 
under way. In parallel, the use of a combination of 
anti-CD3 and anti-CD7 immunotoxins to induce 
long-standing  remission  of  severe  high-grade 
graft-versus-host disease, has been investigated 
(drug  components  have  been  produced  and 
purified, draft protocols and inform consent pre-
pared, phase I/II study has been registered and a 
preclinical evaluation has been realised). A study 
using monkeys to perform cytotoxicity tests has 
been prepared. 

Main findings:

Participants

Ethical  aspects  have  been  monitored  through 
WP4 and further research is ongoing for key so-
cietal, organisation, ethical, regulatory and legal 
issues related to activities performed in RISET.   

A  first  series  of  diagnostic  tests  have  been  de-
fined  and  validated  in-house  in  WP1  (work 
package 1), to identify transplanted patients for 
whom  immunosuppressive  treatment  could  be 
safely minimised or withdrawn. The validation of 
these tests and markers in clinical protocols has 
started, with first samples of tolerance induction 
pilot trials already collected and distributed un-
der strict quality control.  

Basic research performed in WP2 has contributed 
to the characterisation at molecular level of im-
portant actors and mediators of tolerance (mark-
ers of regulatory T cells, tolerogenic DCs). Models 
of  endothelial  cell  damage  to  test  the  effect  of 
endothelial  cell  protection  on  allograft  survival 
were  initiated.  Antibody  therapy  in  human  CD3 
or  CD52  transgenic  animals  was  tested,  with  a 
view to optimising anti-CD3 and anti-CD52 treat-
ment in clinical settings. 

Clinical  trials  have  started.  In  total,  12  patients 
have been included in RISET clinical trials so far. 
Safety,  efficacy  and  biological  tests  have  been 
performed for 10 patients. In January and Febru-

Several  dissemination  supports  have  been  de-
veloped (website, leaflet, newsletter, and presen-
tations to scientific and large public audiences). 
Training  actions  related  to  RISET  activities  and 
research objectives have taken place at local, na-
tional and international level. A feasibility study 
to examine the opportunity of setting up an in-
dustry platform to foster exploitation aspects of 
the project has been performed, and has led to 
recommendations for RISET’s future activities.

Coordinator 

MichelGoldman
Université Libre de Bruxelles
Rue Adrienne Bolland, 8
6041 Charleroi (Gosselies), Belgium 
E-mail:mgoldman@ulb.ac.be

Partners

Hans-DieterVolk
Charite, University Medicine Berlin
Berlin, Germany

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IMMUNOTHERAPY ANd TRANSPLANTATION

M.C.Cuturi
CHU Nantes
Nantes, France

M.DahaandF.Claas
Leids Universitair Medisch Centrum
Leiden, Netherlands

L.Chatenoud
Université René Descartes Paris
Hospital Necker
Paris, France

K.Wood
Oxford University 
John Radcliffe Hospital
Nuffield Department of Surgery, level 6, Headington
Oxford, UK

U.Janssen
Miltenyi Biotec GmbH
Cologne, Germany

H.Waldmann
Sir William Dunn School of Pathology
Oxford, UK

M.G.Roncarolo
San Raffaele Telethon Institute for Gene Therapy
Milan, Italy

R.Lechler
Kings College 
Hodgkin Building, Guy’s Campus
London, UK

R.Rieben
Bern University Hospital
Bern, Switzerland

F.Fandrich
Blasticon Gmb
Kiel, Germany

B.Arnold
Deutsches Krebsforschungszentrum
Heidelberg, Germany

A.Cambon-Thomsen
INSERM U558
Toulouse, France

J.P.Soulillou
CHU Nantes
Nantes, France

O.Vilkicky
Institute for Clinical and Experimental Medicine
Prague, Czech Republic

M.Guillet
TC Land
Nantes, France

U.Kunzendorf
University of Schleswig-Holstein
Kiel, Germany

B.Miranda
Organización Nacional de Transplantes
Madrid, Spain

Y.Reisner
Weizmann Institute of Science
Rehovot, Israel

A.Wendel
Université Konstanz
Konstanz, Germany

N.Schwabe
ProImmune Ltd
Oxford, UK

S.Houard
Henogen
Gosselies, Belgium

NewTherapy_02.indd   223

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New Therapies – Immunotherapy and Transplantation

223

XENOME

Engineeringoftheporcinegenomeforxenotransplantationstudiesinprimates:
asteptowardsclinicalapplication

IMMUNOTHERAPY ANd TRANSPLANTATION






ContractNo 
Projecttype

LSHB-CT-2006-037377
IntegratedProject

ECcontribution  e 987646

Startingdate 1November2006

Duration 60months

Website  www.xenome.eu

Background and objectives:

The ultimate goal of XENOME is to generate the 
necessary  data  to  allow  xenotransplantation  to 
advance towards its initial clinical phase. The data 
generated in this project will encompass both ef-
ficacy and safety aspects of xenotransplantation. 
Tools  that  will  be  used  to  reach  this  ambitious 
objective  include  state-of-the-art  biomolecular 
technologies and in vivo models. 

XENOME  aims  to  produce  a ‘super-engineered’ 
pig,  i.e.  a  pig  with  a  newly  generated  genotype 
that  will  improve  the  efficacy  and  safety  pro-
file  of  xenotransplantation.  Assessments  of  ef-
ficacy  will  first  take  advantage  of  existing  pig 
lines expressing human complement regulators, 
thrombomodulin  (TM)  and  knock-out  for  Δ-Gal 
transferase  (Δ-GalT  KO).  Using  the  most  suitable 
background,  further  engineering  of  the  pig  ge-
nome will be undertaken. Additional transgenes 
able  to  control  immune  responses,  endothelial 
cell activation and subsequent microangiopathy, 
will be added. 

The  ultimate  pig  strain  will  thus  combine  the 
already  available  background  with  novel  mol-
ecules  exhibiting  anticoagulant  and  immuno-
suppressive  properties.  In  addition,  an  effective 
immuno-suppression  regimen  will  be  defined, 
and new pharmaceutical agents will be tested. A 
strong safety framework will also be established, 
that may allow, at some stage, the progression of 
xenotransplantation  into  the  clinic. This  will  en-

tail  the  development  of  technologies  enabling 
the  timely  diagnosis  of  infection,  the  design  of 
a  safety  plan  for  an  efficacious  containment  of 
an untoward infectious event, the breeding of a 
herd  of ‘clean’  source  pigs,  and  the  provision  of 
safety-related  data  derived  from  long-term  in 
vivo  studies  in  primate  xenograft  recipients.  Fi-
nally,  the  project  will  also  offer  a  strong  ethical, 
social  (especially  regarding  public  communica-
tion)  and  regulatory  framework  for  xenotrans-
plantation research (and possibly for clinical ap-
plication too).

Xenotransplantation  addresses  the  growing 
problem of the shortage of human organs, which 
prevents many patients with terminal organ fail-
ure from receiving a human organ. As a result of 
improved  medical  and  technological  interven-
tions in the field of transplantation, an increasing 
number of people are now short-listed for trans-
plants.  The  waiting  lists  continue  to  grow  due 
to a limited organ supply; it is, for instance, esti-
mated that approximately 45 000 people across 
the EU are currently on waiting lists for a kidney 
transplant.  
The  fundamental  objectives  of  this  project  are 
presented below:

•  Bring  the  EU  back  to  the  forefront  of 
xenotransplantation research, a field with 
a huge potential for the treatment of end-
stage organ failure. At the end of the pro-
gramme, XENOME will deliver a European 
‘product’ for xenotransplantation. This will 
consist  of  a  ‘super-engineered’  pig  with 

224

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IMMUNOTHERAPY ANd TRANSPLANTATION

7
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long-term  survival 

European intellectual property rights.
•  Generate  in vivo  efficacy  data:  experi-
mental evidence that the ‘engineered’ pig, 
combined  with  new  immunosuppressive 
reagents,  allows 
in 
non-human primates. 
•  Assess  new  immunosuppressive  regi-
mens  and  agents  for  the  efficient  inhibi-
tion of complement activation and acute 
vascular rejection.
•  Gain  a  better  understanding  of  the 
physiology  of 
long-term  transplanted 
kidney  xenografts  in  the  pig-to-primate 
model.
•  Establish  the  necessary  safety  frame-
work that would allow the progression of 
xenotransplantation into the clinic. 
•  Provide a strong ethical, social, educa-
tional (especially apropos communication 
with  the  public)  and  regulatory  frame-
work,  within  which  xenotransplantation 
research (and possibly clinical application) 
should take place.

Approach and methodology:

Addressing safety and the efficacy of xenotrans-
plantation is the major endeavour of this initia-
tive.  Novel  constructs  that  could  confer  advan-
tages to the xenograft will be passed on to the 
pig-engineering team whose principal objective 
will  be  to  modify  the  pig  characteristic  profile 
according  to  the  desired  traits.  The  biomolecu-
lar tools applied will allow complete abrogation 
(knock-out),  reduced  expression  (knock-down), 
or over-expression of candidate genes of interest 
in porcine endothelial cells (transgenesis). 

The  pig-engineering  team  in  XENOME  will  en-
sure that the in vitro findings generated are ade-
quately utilised and translated to in vivo work, re-
sulting in novel and stable pig lines. It will also be 
part of the responsibilities of the same team to 
ensure that animals are appropriately screened, 
bred and provided in sufficient number to gener-
ate the proof-of-concept data in in vivo primate 
studies.  Indeed,  when  new  lines  of  engineered 

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New Therapies – Immunotherapy and Transplantation

22

 
 
 
 
 
 
IMMUNOTHERAPY ANd TRANSPLANTATION

XENOME

pigs  are  made  available  to  this  network,  the  ef-
ficacy and safety of the organs will be tested by 
transplanting them into primates. 

All the activities undertaken will be tightly moni-
tored  and  supervised  by  an  ethical  and  legal 
team,  whose  responsibilities  will  include  ensur-
ing  that  all  the  initiatives  conducted  within  the 
scope of this programme are fully compliant with 
existing European ethical and legal frameworks. 
Furthermore,  this  team  will  target  the  need  to 
bridge the gaps between laboratories and soci-
ety, and between scientists, citizens and institu-
tions,  both  by  creating  and  maintaining  a  con-
tinuous  communication  flow  with  the  scientific 
partners, as well as by establishing a connection 
with the general public. This will be made possi-
ble via diverse means, such as the preparation of 
videos and leaflets, and the organisation of open 
laboratory workshops. Public consultation activi-
ties will also be conducted. 

Expected outcome:

XENOME  is  expected  to  generate  the  neces-
sary set of data that may allow the transition of 
xenotransplantation from the current preclinical 
phase to its initial clinical phase, within the next 
10 years. This is an ambitious goal and the data 
that will be generated during the five- year dura-
tion of this project will address both the efficacy 
and safety aspects of xenotransplantation.

Coordinator

EmanueleCozzi
Azienda Ospedaliera di Padova
Ospedale Giustinianeo
Via Giustiniani, 2
Padova, Italy
E-mail: emanuele.cozzi@unipd.it

Partners

Jean-PaulSoulillou
ITERT
Nantes, France 

PierreGianello
Université Catholique de Louvain
Leuven, Belgium

CarlosRomeo-Casabona
Universidad del Pais Vasco
Bilbao, Spain

MarialuisaLavitrano
University of Milan
Milan, Italy

RainerdeMartin
Medical University of Vienna
Vienna, Austria

YasuhiroTakeuchi
University College London
London, UK

HeinerNiemann
Institute for Animal Breeding, Federal Agricultural 
Research Centre
Neustadt, Germany

LindaScobie
University of Glasgow
Glasgow, UK

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IMMUNOTHERAPY ANd TRANSPLANTATION

CesareGalli
Consorzio per l’incremento Zootecnico srl
Cremona, Italy

MohamedR.Daha
Leiden University Medical Centre
Leiden, Netherlands

JiriHejner
Institute of Molecular Genetics, Academy of Sciences of 
the Czech Republic
Prague, Czech Republic

ReinhardSchwinzer
Hannover Medical School
Hannover, Germany

PaoloSimoni
University of Padua
Padua, Italy

RogerBarker
Cambridge Centre for Brain Repair
Cambridge, UK

JohannesRegenbogen
GATC Biotech AG
Konstanz, Germany

ErmannoAncona
Consorzio per la Ricerca sul trapianto d’Organi
Padova, Italy

OlleKorsgren
Corline Systems AB
Uppsala, Sweden

GuilhermeDeOliveira
Biomedical Law Centre
Coimbra, Portugal

MariachiaraTallachini
Università Cattolica S.C. Milano
Milan, Italy

MiguelCheParreiraSoares
Instituto Gulbenkian de Ciência
Oeiras, Portugal

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New Therapies – Immunotherapy and Transplantation

227

CLINT

Facilitatinginternationalprospectiveclinicaltrialsinstemcelltransplantation

IMMUNOTHERAPY ANd TRANSPLANTATION

ContractNo 
Projecttype

LSHB-CT-2007-037662
SpecificSupportAction

ECcontribution  e 00000

Startingdate 1April2007

Duration 24months






severely curtailed by the recently introduced re-
quirement  to  carry  out  these  studies  in  accord-
ance with the EU Directive on Clinical Trials. 

legislation  have  rendered 

Although  introduced  with  laudable  intentions, 
the  effect  of  the  directive  has  been  to  increase 
the  resources  and  therefore  the  expense  in-
volved  in  clinical  trials,  whilst  at  the  same  time 
national  differences  in  the  interpretation  of  the 
new 
international 
studies extremely difficult. The objective of CLINT 
is to support the EBMT to further develop the in-
frastructure necessary to perform academically-
initiated international prospective studies in SCT 
throughout  Europe. This  will  hasten  the  evalua-
tion  of  new  treatment  strategies  and  improve 
the  outcome  for  European  citizens.  The  CLINT 
consortium will be assisted in this endeavour, by 
advice  provided  from  a  similar  initiative  in  the 
United States.

Background and objectives:

Autologous  and  allogeneic  stem  cell  transplan-
tation (SCT) is the treatment of choice for many 
haematological diseases. Within healthcare pro-
vision, SCT is not only one of the most costly but 
it is also one of the most risky procedures for pa-
tients, with transplant related mortalities of up to 
50%.  SCT  has  always  been  on  the  cutting  edge 
of translational medicine, and over the past dec-
ade there have been many changes to the way 
in which transplant is performed, such as the in-
troduction of new drugs and technologies. While 
these innovations have the potential to improve 
patient outcome, they can also increase the cost 
considerably. At the same time, many new devel-
opments have emerged with respect to targeted 
drug therapies; leave supportive care in these dis-
eases and therapies may replace or delay trans-
plant  for  some  patients.  These  new  treatments 
are also expensive and require urgent evaluation. 
It is essential that they and SCT are used wisely 
and economically. 

There  has  always  been  willingness  for  the  SCT 
community to critically evaluate the role of trans-
plants,  as  is  exemplified  by  the  transmission  of 
outcome data from individual centres to a cen-
tral database held by the European Bone Marrow 
Transplant Group (EBMT) for further analysis and 
reporting. SCT physicians have also been enthu-
siastic exponents of clinical trials, and are ready 
to test new hypotheses, and to compare SCT with 
other treatments. Their ability to do this has been 

228

New Therapies – Immunotherapy and Transplantation

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IMMUNOTHERAPY ANd TRANSPLANTATION

Coordinator

JaneApperley
Imperial College
Department of Haematology
Hammersmith Hospital
Ducane Road
London W12 0NN, UK
E-mail: j.apperley@imperial.ac.uk

Partners

FionaMacDonald
European Group for Blood and Marrow Transplantation
Barcelona, Spain

DorisSchroeder
University of Central Lancashire
Preston, UK

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New Therapies – Immunotherapy and Transplantation

229

TRIE

TransplantationresearchintegrationacrossEurope

IMMUNOTHERAPY ANd TRANSPLANTATION

ContractNo 
Projecttype

LSHB-CT-2007-03740
SpecificSupportAction

ECcontribution  e 424732

Startingdate 1March2007
Duration 24months






Background and objectives:

The  primary  objectives  of  the  project  are  out-
lined below: 

1. To  identify  current  opportunities  and  chal-
lenges  in  the  field  of  transplantation  research, 
focusing  on  themes  common  to  cell  and  solid 
organ  transplantation.  In  order  to  achieve  this 
overall  objective,  the  following  sub-objectives 
are required: 

•  The establishment of a respected advisory 
council which includes the following: 
- 
recognised  scientists  in  the  field  of 
transplantation  research,  selected  on  the 
basis of ‘peer recognition’,
representatives  of  the  key  organisa-
- 
tions involved in transplantation research 
in Europe, i.e. the European Society for Or-
gan Transplantation (ESOT) and the Euro-
pean Group for Blood and Marrow Trans-
plant Society (EBMT),
- 
representatives of relevant EU-funded 
research projects in the field of transplan-
tation  (e.g.  RISET,  Allostem,  Alliance-O, 
DOPKA etc),
-  a  representative  of  the  European 
Agency for Evaluation of Medicinal Prod-
ucts (EMEA),
- 
the field (both big pharmas and SMEs);
•  To  identify  common  opportunities  and 
challenges  on  different  transplantation 
research themes, through discussions and 

representatives  of  industries  active  in 

consultations  with  working  groups  and 
members of the advisory council:
-  biomarkers  and  pharmacogenomics, 
to tailor immunosuppression,
-  how to promote living donation
- 
immunotherapies,
- 
munomonitoring tests,
- 
tion medicine;

regulatory aspects of novel cell-based 

standardisation  and  validation  of  im-

training  programmes  in  transplanta-

2. To prepare a set of recommendations regard-
ing priority actions to be implemented, so as to 
foster  the  integration  of  research  activities  on 
themes  common  to  cell  and  solid  organ  trans-
plantation. In order to achieve this overall objec-
tive, the following sub-objectives are required: 

to  form  working  groups  on  different 
• 
research  themes,  to  facilitate  discussion 
and consultation in order to reach a con-
sensus on existing ‘gaps’;
• 
to prepare detailed recommendations 
on optimal measures to address the gaps;
•  based  on  the  output  of  the  working 
groups, to prepare a consensual position 
paper  that  will  present  in  an  articulated 
manner the challenges, opportunities and 
recommendations in terms of priority ac-
tions  to  integrate  research  activities  on 
cell and solid organ transplantation.

3.Organisation of a public ‘dissemination’ event 
in  close  collaboration  with  media  experts  and 

230

New Therapies – Immunotherapy and Transplantation

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IMMUNOTHERAPY ANd TRANSPLANTATION

patients’ representatives. This event would have 
the following two sub-objectives:

•  project  coordinators  and  participants 
would  present  concrete  results  of  past 
and current research efforts — supported 
namely  by  the  EU  Commission  —  in  the 
field of cell and organ transplantation;
• 
put of TRIE with stakeholders.

to present, discuss and debate the out-

Coordinator

MichelGoldman
Université Libre de Bruxelles
Institute for Medical Immunology
Rue Adrienne Bolland, 8
B-6041 Charleroi (Gosselies), Belgium
E-mail: mgoldman@ulb.ac.be

Partners

KathrynWood
University of Oxford
Nuffield Department of Surgery - Immunology
Oxford, UK

AlejandroMadrigal
The Antony Nolan Trust
London, UK

BlancaMiranda
Organización Nacional de Trasplantes 
Madrid, Spain

SiobhànMcQuaid
Abu International Project Management Ltd
Dublin, Ireland

NewTherapy_02.indd   231

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New Therapies – Immunotherapy and Transplantation

231

 
 
NEW THERAPIES

232

New Therapies – Index of Projects

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NEW THERAPIES

INdEX OF PROJECTS

STEMDIAGNOSTICS
STEM-HD
STEMS
STEMSTROKE
STROKEMAP
SyntheGeneDelivery
THERADPOX
THERAPEUSKIN
THERAVAC
TherCord
THOVLEN
TRIE
UlcerTherapy
XENOME
ZNIP

217
72
64
61
67
138
107
44
169
30
104
230
37
224
115

AlloStem
ANGIOSKIN 

AutoCure
BacAbS


BACULOGENES
BetaCellTherapy
BMC
CELLAID
CLINIGENE
CLINT
COMPUVAC 

CONSERT

CRYSTAL 

DC-THERA
DC-VACC

DENTRITOPHAGES
EPISTEM 

EPI-VECTOR
EuroSTEC

EuroStemCell
GenomesToVaccines
GENOSTEM
GIANT
HEPACIVAC 
ImprovedPrecision
IndustryVectorTrain
INNOCHEM
INTHER
InVivoVectorTrain
Magselectofection
MimoVax
MOLEDA
Myoamp
NEUROscreen
OsteoCord
Pharma-Planta
PolExGene
RESCUE
RIGHT
RISET
SAGE
SC&CR
Skintherapy
SNIPER

155
144
204
189
102
47
202
214
85
228
181
92
80
159
165
173
32
129
52
17
177
22
98
186
121
150
209
124
147
135
193
141
77
75
27
196
133
69
110
221
200
57
41
118

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New Therapies – Index of Projects

233

INdEX OF
COORdINATORS

NEW THERAPIES

NETWORK OF EXCELLENCE

Austyn, Jonathan M (DC-THERA)  
Cohen-Haguenauer, Odile (CLINIGENE) 

163
 89

INTEgRATEd PROJECTS

Cortese, Riccardo (HEPACIVAC)  
Cozzi, Emanuele (XENOME) 
Feitz, Wouter (EUROSTEC)  
Fischer, Rainer (Pharma-Planta)  
Goldman, Michel (RISET)  
Jorgensen, Christian (GENOSTEM)  
Klareskog, Lars (AutoCure) 
Klatzmann, David (COMPUVAC)  
Ma, Julian
(Pharma-Planta, scientific coordinator)  
Madrigal, Alejandro (AlloStem)  
Maitland, Norman J. (GIANT) 
Meyer, Thomas F. (RIGHT)  
Mantovani, Alberto (INNOCHEM)  
Pipeleers, Daniel (BetaCellTherapy) 
Schenk-Braat, Ellen
(GIANT, scientific coordinator)  
Smith, Austin (EuroStemCell) 
Vandenabeele, Peter (EPISTEM)  
Wagemaker, Gerard (CONSERT)  

SPECIFIC SUPPORT ACTION

Apperley, Jane (CLINT)  
Goldman, Michel (TRIE)  
Mezzina, Mauro (InVivoVectorTrain)  
(IndustryVectorTrain)  
Radbruch Andreas (CELLAID)  

188
 226

196
222
2
 207
18

198
17
 101
113
213
 0

101
 21
3
96

229
231
149
11
216

234

New Therapies – Index of Coordinators

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INdEX OF

COORdINATORS

SPECIFIC TARgETEd RESEARCH

NEW THERAPIES

Allsopp, Tim (NEUROscreen)  
7
Bartholeyns, Jacques (DENDRITOPHAGES)   176
 140
Bigot, Yves (SyntheGeneDelivery) 
180
Buus, Søren (Genomes To Vaccines)  
60
Capogrossi, Maurizio C. (SC&CR)  
192
Daura, Xavier (BacAbS)  
 219
Dickinson, Anne (STEMDIAGNOSTICS) 
106
Epstein, Alberto (THOVLEN)  
 29
Genever, Paul (OsteoCord) 
82
Hescheler, Jürgen (CRYSTAL)  
Hook, Lilian
(NEUROscreen, scientific coordinator)  
Hovnanian, Alain (THERAPEUSKIN)  
Izsvák, Zsuzsanna (INTHER) 
Jackson, Dean A. (EpiVector)  
Kadouche, Jean (BMC)  
Kokaia, Zaal (StemStroke) 
Krauss, Stefan (SNIPER)  
Krauss, Stefan (ZNIP)  
Leclerc, Claude (THERAVAC)  
Lusky, Monika (THERADPOX)  
Martin, John (BACULOGENES) 
Mattner, Frank (MimoVax)  
Meneguzzi, Guerrino (Skintherapy) 
Mir, Lluis M. (ANGIOSKIN)  
Mouly, Vincent
(myoamp, scientific coordinator)  
Onteniente, Brigitte (STEMS) 
Peschanski, Marc (STEM-HD)  
Plank, Christian (Magselectofection)  
Privat, Alain (RESCUE) 
Rosenecker, Joseph (Improved Precision)  
Schacht, Etienne (PolExGene) 
Scherman, Daniel (MOLEDA)  
Schillberg, Stefan (SAGE)  
Verfaillie, Catherine (STROKEMAP)  
Zambruno, Giovanna (Ulcer Therapy) 

7
46
 128
132
203
 63
120
117
172
109
 103
19
 43
14

79
 66
74
137
 71
123
 134
143
201
68
 40

NewTherapy_02.indd   235

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New Therapies – Index of Coordinators

23

NewTherapy_02.indd   234

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NewTherapy_02.indd   235

11/8/07   10:50:31 AM

NewTherapy_02.indd   237

11/8/07   10:50:31 AM

How to obtain EU publications 
Our priced publications are available from EU Bookshop (http://bookshop.europa.eu/), where you 
can place an order with the sales agent of your choice. 

The Publications Office has a worldwide network of sales agents. You can obtain their contact 
details by(cid:1)sending a fax to (352) 29 29-42758. 

NewTherapy_02.indd   236

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NewTherapy_02.indd   239

11/8/07   10:50:31 AM

European Commission —  
Directorate-General for Research Life Sciences, Genomics and Biotechnology for  Health 

NEW THERAPIES — EU-supported Research in Genomics and Biotechnology  for Health 
Sixth Framework Programme (2002-2006) 

EUR 22841 

Luxembourg: Office for Official Publications of the European Communities 

2007 —  236 pp. —  17.6 x 25 cm 

ISBN:  978-92-79-05562-1 

Catalogue number:  KI-NA-22841-EN-C 

Price (excluding VAT) in Luxembourg: 25.00 EUR  

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NewTherapy_02.indd   240

11/8/07   10:50:31 AM