Research on rare diseases follows a
long and complex pathway (1)
Identification of a clinical phenotype
Mendelian
Genetics
Gene identification
Validation
cohort
clinical expertise
databases
technical platforms
Genotyping
Sequencing
«Variants»
(same phenotype without mutation)
Genotype/phenotype
correlation ?
DIAGNOSIS
Gene modifiers
PROGNOSIS
Pathophysiology
Cell studies
Gene expression analysis
Animal models
In vivo studies
Bioinformatics,
cell imaging…
Animal transgenic
facilities
Phenotyping facilities
(Imaging…)
Research on rare diseases follows a long
and complex pathway (2)
Therapeutic research
Compounds libraries
Screening
Industrial Collaborations
Targets
Pathophysiology
Screening of
molecules
Class of
molecules
Biological
molecules
Cell and gene
therapy
in vitro
Vectorology
large animals facilities
in vivo
Animal models
Clinical trials
specific methodology
THERAPEUTICS
The "reference center" for primary immunodeficiencies
(PID). Necker University Hospital Paris
PID diagnosis
laboratory
Dpt of
Biotherapy
research
Faculté
deU550
Médecin
e
U429
UIH Pediatrics
Infectiology,
immunology
adults
Data base center
SCID diseases
Fatal conditions early in life
X-linked or autosomal recessive inheritance
T
HSC
Myeloid
compartment
THYMUS
T
B
1970s
SCID diseases
1985
NK
Abnormal thymus in SCID
HSC
CLP
Myeloid
compartment
Control
THYMUS
SCID
CD8
CD4
B
X-linked SCID
1985-1995
3
22.
2
IL-15
1
p
21.
3
NK
gc deficiency
2
1
11.
11.
12
4
3
23
22
21
1
1
2
TIMP
DXS255
DXS14
DXZ1
DXS159
13
PGK1
DXS447
1
21.
2
q
23
XL-SCID
Déficit immunitaire
combiné sévère
gc gene
T
CD4
IL-7
HSC
T
CD8
DXS3
3
22.
CLP
B
DXS178
1
2
3
DXS94
24
DXS17
25
DXS42
IL-4
IL-2
IL-7
IL-9
IL-15
IL-21
DXS37
26
IL-2Rb
IL-2Ra
27
28
X
gc
IL-4Ra
gc
IL-7Ra
gc
IL-9R
gc
IL-2Rb
IL-15Ra
gc
IL-21R
gc
T-B-SCID
p
Ly NK
T gd
Pro T
Chr. 10
T CD8
T CD4
Precursor
Pre-B
B
Pro B
"T-B-SCID"
B
IgG, A, E
B
IgM
q
100
Genomic deletions / Splice site mutations
1-3
5-6
4
5
b-Lact
N
M1T
S32C R81X
H35D
Artemis
5-8
G118V
G135E
10-12
9 10
% Survival
3
11
b-CASP
del. 1bp
R191X
del. 4bp
Y199X
SCIDA
C-ter
10
1
C
Control
RS-SCID
RS-SCID + Arte
del. 7bp
del. 17bp
ins. 2bp
0
1
Gy
2
1990-2001
3
T-B-SCID
Artemis KO
Spleen
Thymus
5.7
76.1
61.8
8.7
4.5
WT
0.2
0.3
TCR and BCR genes rearrangements
during T and B cell differentiation
v
0.1
Cleavage
KO
1.8
CD8
13.0
RSS
j
Rag-1-Rag-2
v
IgM
CD4
Hairpin
coding ends
RSS
B220
j
Ku70-Ku80
DNA-PKcs
Hairpin opening
and processing
Artemis
j
v
End joining
Excised
DNA
XRCC4, ligase 4
Generation of coding joints
2001-2004
SCID diseases
1.Prevention of cell apoptosis
DNA replication (purin metabolism)
ADA
2005
2.gc cytokine-dependent signals
gc, JAK-3, IL7Ra
NK
4.Pre TCR/TCR signalling)
CD45, CD3d,
HSC
CD8
CLP
CD4
Myeloid
compartment
THYMUS
3.V(D)J recombination
Rag-1/-2, Artemis
B
More diseases to be described…
Molecular analysis of SCID conditions, what for ?
1. Basic science: lymphocyte development
2. Molecular diagnosis/genetic counseling
3. Specific therapies: ADA enzyme substitution, gene therapy
4. Design of new drugs:
. ADA deficiency  lymphotoxic role of deoxyadenosine
 2 chloro deoxyadenosine, a cytotoxic drug used
in the treatment of lymphomas
. gc/JAK-3 deficiencies  role of JAK-3 in lymphocyte
development/survival/function
 JAK-3 inhibitor as a new immunosuppressive agent used
in transplantation…
Schematic representation of a major
signaling pathway activated by IL-2
JAK-3 inhibitor
GAS
Description of multiple SCID variants
Three examples:
 Omenn's syndrome
 Hypomorphic mutations of Artemis
 Revertant in XL-SCID
Omenn’s Syndrome: hypomorphic mutations in Rag-1/2
emergence of autoreactive T cell clones
skin
CD3 staining
1
1043
K86Fs.
gu
t
G720S
R1010H
R511H
L732F
K1029E
R404W
R404Q
D832N
S875Fs.
Hypomorphic mutations of Rag-1
Defective central T cell tolerance
Patient
Control
T173Fs.
Y333•
Oligoclonal T cell expansion
Hypomorphic mutations ofArtemis
associated with B cell lymphomas
b-Lact
C-ter
b-CASP
“Catalytic domain”
“Regulatory domain”
T432 fs
D451 fs
Hypomorphic mutations
Phenotype ?
Occurrence of B cell lymphomas
CD20
Ki67
Role of Artemis as a genome caretaker
An atypical case of XL-SCID
gc deficiency
NK
NK
Reverse mutation
T
CD4
HSC
T
CD8
B
Absence of T cells
Absence of NK cells
T
CD4
gc
gc
gc
T
CD8
gc
gc
B
T cell counts = 50 % of control
memory phenotype
Repertoire diversity generated from
one precursor (revertant cell) ?
Revertant analysis
• 1 T cell precursor 
1 x 103 TCRVB clones
 10 to 11 cell division cycles
~ 1 % of T cell repertoire diversity
A form of natural gene therapy !
Probability of survival in SCID patients after
hematopoietic stem cell transplantation according
to donor-recipient compatibility
1968: the 1st successful HSCT was achieved in a patient with SCID
HLA id. sibling
T cells/µl
Survival %
3,000
HLA haplo id. parent
Homeostatic expansion of
donor mature T cells
HLA id.
Haplo id.
2,000
New T cell
ontogeny
1,000
0
1
2
3
4
6
12
months
Months
European registry - 464 pts
Persisting severe immunodeficiency
Lymphopoiesis in SCID patients pre/post
hematopoietic stem celltransplantation (HSCT)
Pre HSCT
Early post HSCT
Donor
Late post HSCT
NK
T
HSC
Exhaustion of
precursors
T
Immunodeficiency !
Myeloid cells
No donor cells in bone marrow
Occurrence of severe chronic skin HPV disease
in SCID patients after HSCT
gc, JAK3
deficiencies
Occurrence of severe chronic skin HPV disease
in SCID patients after HSCT
• A significant problem with no simple solution
• An unexpected role for gc/JAK-3 cytokine receptor
signaling pathways in defense of keratinocytes
against papilloma virus
 New therapeutics ?
Principle of ex vivo gene therapy
of the SCID-X1 condition
T Lymphocytes development after
gene therapy of SCID-X1
9000
8000
P4
7000
*
6000
T
Lymphocytes/µl
P8
5000
*
P9
4000
3000
P2
P7
2000
P5
P6
1000
*
P1
P10
0
0
10
20
30
40
Months after Gene Therapy
50
60
70
High proliferation rate enables T cell diversification
from a few precursors
Transduction
1 clone
Multiple T cell clones
1 integration site,
diverse TCR
CLP
gc-dependent
proliferation
Hematopoietic
stem cell
Myeloid cells
TCR
rearrangements
As in the reversion case, oligoclonal lymphopoiesis
 polyclonal repertoire
Monoclonal gd T cell proliferation P4
T Lymphocytes/µl
1000000
100000
10000
1000
100
P4
10
1
0
5
10 15 20 25 30 35 40
46, XY, 6q21;13qter
• Monoclonal g d T cell clone
Serious adverse effect caused by retroviral
insertional mutagenesis in a protooncogene
44,218
MFG gc
P5
46,229
MFG gc
1
P4
54,614
66,467
66,867
71,646
76,883
2
3
4
5
6
Enhancer activity
LMO-2 mRNA
2. 3 kb
Aberrant expression ---> clonal proliferation
1
2
3
4
5
1077
6
1513
T cell clone
Clonal lymphoproliferation
Synergistic effect with the
gc transgene
Conclusion
Clinical benefit with sustained efficacy
• 6 additional successfully treated patients in UK (A.Thrasher)
 extension to other SCID ?
 ADA deficiency, Rag-1, Rag-2, Artemis deficiencies…
•
•
But… a serious adverse effect
 vector modifications
Vector modifications
LTR

gc
Promoter
LTR
IRES suicide
gene
 U3
Self inactivated LTR
loss of the enhancer
effect
Principles for research on rare diseases
• Clinical centers of expertise in limited numbers
- well studied cohorts of patients
• International collaborative effort
• Multidisciplinary approach
• Impact  - basic science
. lymphocyte development
- knowledge and therapy of more common diseases
. pathophysiology of papilloma virus disease,
. hematopoietic stem cell transplantation,
. gene therapy
- new drugs
. treatment of lymphomas, prevention of graft rejection
An European institute
supporting research on rare diseases
• To organize disease-reference centers (national/regional),
in an european network
• To build a network of platforms available for research projects
on rare diseases (genomics,animal house, post genomics,
molecular screening… )
• To foster collaboration with pharmaceutical companies.
- example: the ERDITI initiative
ERDITI: European Rare Diseases Therapeutic
Initiative
A public/private partnership

To provide academic teams with an access to a variety
of compounds from pharmaceutical companies
- based on pathophysiological hypotheses

To provide a collaboration process between academic teams
and pharma partners
Under the European Science Foundation
Coordinated by GIS – Institut des maladies rares
Partners and supporting institutions
Pharmaceutical partners
Supporting institutions
 Aventis
 Austria, Medical University Vienna
 Glaxosmithkline
 Belgium, FNRS - Fonds national de la
 Roche
 Servier
Recherche Scientifique
 Croatia, Academy of Science ans Arts
 Denmark, Medical research council
 France,
CNRS and Inserm
 Germany, DLR Project managment
Organisations – Health research
 Netherlands, ZonMw, Steering
committee on Orphan Drugs
 Spain, Rare diseases Institute (IIER)
 Slovakia, Academy of sciences
A charter of collaboration
•
Partnership based on a charter of collaboration
•
The charter has 3 sections:
- working procedure
- standard material transfer agreement (MTA)
- standard intellectual property rights agreement
•
Assessment by a scientific advisory board
- analysis of the accuracy of the proposal
- advices
www.erditi.org
www.institutmaladiesrares.net