research on rare diseases follows a long and complex pathway (1)
DESCRIPTION
Mendelian Genetics. Gene identification. Genotyping Sequencing. «Variants» (same phenotype without mutation). DIAGNOSIS. Validation. Genotype/phenotype correlation ?. PROGNOSIS. Gene modifiers. Pathophysiology. Bioinformatics, cell imaging…. Cell studies Gene expression analysis. - PowerPoint PPT PresentationTRANSCRIPT
Research on rare diseases follows a long and complex
pathway (1)
Identification of a clinical phenotype
Mendelian Genetics
Genotyping Sequencing
Gene identification
cohortclinical expertisedatabasestechnical platforms
DIAGNOSIS«Variants»(same phenotype without mutation)
Genotype/phenotype correlation ?
PROGNOSIS
Gene modifiers
Validation
Cell studiesGene expression analysis
PathophysiologyBioinformatics, cell imaging…
Animal transgenic facilities Phenotyping facilities (Imaging…)
Animal models
In vivo studies
Therapeutic research
Research on rare diseases follows a long and complex pathway (2)
Compounds libraries
Screening
Industrial Collaborations
in vitroin vitro
Screening of Screening of moleculesmolecules
Targets
Class of Class of moleculesmolecules
Biological Biological moleculesmolecules
Pathophysiology
in vivoin vivo
Animal models
Vectorology
large animals facilities
Cell and gene Cell and gene therapytherapy
THERAPEUTICSClinical trials specific methodology
The "reference center" for primary immunodeficiencies (PID). Necker University
Hospital Paris
Infectiology, Infectiology, immunology immunology
adultsadults
FacultFacultéédedeMédeciMédecinene
Dpt of Dpt of BiotherapyBiotherapy
PID diagnosislaboratory
UIH UIH PediatriPediatricscs
Data base Data base centercenter
U429
U429
U550U550
researchresearch
SCID diseases
1970s
Fatal conditions early in life X-linked or autosomal recessive inheritance
THYMUSMyeloid compartment
T
T
B
HSC
THYMUSMyeloid compartment
SCID diseases1985
B
HSC CLP
CD8
CD4
NK
ControControll
SCIDSCID
Abnormal thymus in Abnormal thymus in SCIDSCID
X-linked SCID
IL-2IL-2 IL-4IL-4 IL-7IL-7 IL-15IL-15 IL-21IL-21
cIL-2RIL-2R
IL-4R c c c cIL-7R IL-2RIL-15R
IL-9IL-9
cIL-9R IL-21R
c deficiencyc deficiency
TTCD4CD4
NKNK
HSCHSC
BB
TTCD8CD8
CLPCLP
IL-15IL-15
IL-7IL-7
1985-1995
p
q
X
11.
21.
22.
11.12
13
21.
22.
23
24
25
26
27
28
1
3
123
12
2122233
1
2
3
1
2
3
4
1
TIMPDXS255DXS14DXZ1
DXS159
PGK1
DXS3
DXS178
DXS94
DXS17DXS42
DXS37
2
Déficit immunitairecombiné sévère
DXS447
XL-SCIDXL-SCIDc genec gene
IgM
IgG, A, E
B
BPre-B
Ly NK
T
T CD8
T CD4
Pro T
Pro B
Precursor
"T-B-SCID"
B
Chr. 10
p
q
10
ins. 2bpS32CR81X
G118V
G135E Y199XSCIDA
R191X
del. 1bpdel. 4bp del. 17bp
del. 7bp
1-3
3
5-6 10-12
5-8
Genomic deletions / Splice site mutations
4 5 9 11
M1T
H35D
N C-Lact -CASP C-ter
ArtemisArtemis
T-B-SCID
1
10
100
0 1 2 3Gy
% S
urv
ival
RS-SCIDRS-SCID + Arte
Control
1990-2001
WT
KO
Thymus
1.8
0.2 0.3
CD8
CD4
8.7
5.7 76.1
Spleen
13.0
0.1
4.5
61.8
B220
IgM
Artemis KO
Hairpin Hairpin opening opening
and and processingprocessing vv jj
Ku70-Ku80Ku70-Ku80
ArtemiArtemiss
DNA-PKcsDNA-PKcs
XRCC4, ligase 4XRCC4, ligase 4
End End joiningjoining
Generation of coding Generation of coding jointsjoints
Excised Excised DNADNA
vv RSSRSS jj
Rag-1-Rag-2Rag-1-Rag-2CleavageCleavageHairpinHairpin
coding endscoding ends
vv jj
RSSRSS
TCR and BCR genes rearrangements during T and
B cell differentiation
T-B-SCID
2001-2004
THYMUS
CLP
NK
CD8
B
2.c cytokine-dependent signals
cc, JAK-3, IL7RaJAK-3, IL7Ra
3.V(D)J recombinationRag-1/-2, Artemis
1.Prevention of cell apoptosisDNA replication (purin
metabolism)ADAADA
HSC
Myeloid compartment
CD4
4.Pre TCR/TCR signalling)CD45, CD3,
SCID diseases 2005
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
therapy4. Design of new drugs:
. ADA deficiency lymphotoxic role of deoxyadenosine 2 chloro deoxyadenosine, a cytotoxic drug used
in the treatment of lymphomas. c/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 JAK-3 inhibitorinhibitor
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
1 1043
T173Fs.
Y333•
R404WR404Q
L732F
D832N
S875Fs.
R1010HK86Fs. G720S
K1029ER511H
Hypomorphic mutations of Rag-1Hypomorphic mutations of Rag-1
CD3 stainingskin
gut
Oligoclonal T cell expansionOligoclonal T cell expansion
Patient
Control
Defective central T cell Defective central T cell tolerancetolerance
T432 fs
D451 fs
“Catalytic domain” “Regulatory domain”
-Lact -CASP C-ter
Hypomorphic mutationsPhenotype ?
Role of Artemis as a genome caretaker
Hypomorphic mutations ofArtemis
associated with B cell lymphomas
CD20CD20 Ki67Ki67
Occurrence of B cell lymphomas
An atypical case of XL-SCID
Absence of T cellsAbsence of T cellsAbsence of NK cellsAbsence of NK cells
T cell counts = 50 % of controlT cell counts = 50 % of controlmemory phenotypememory phenotypeRepertoire diversity generated Repertoire diversity generated from one precursor (revertant from one precursor (revertant cell) ?cell) ?
ccHSCHSC
c deficiencyc deficiency
TTCD4CD4
NKNK
BB
TTCD8CD8
cc
cccc
BB
Reverse Reverse mutationmutation
TTCD4CD4
TTCD8CD8
NKNK
cc
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
HLA id. sibling HLA id. sibling
HLA haplo id. parentHLA haplo id. parent
Months Months
Surv
ival %
Surv
ival %
European registry - 464 European registry - 464 pts pts
months
T cells/µl
1 2 3 4 6 12
0
1,000
2,000
3,000 HLA id. Homeostatic expansion of donor mature T cells
Haplo id.
New T cell ontogeny
Persisting severe Persisting severe immunodeficiencyimmunodeficiency
1968: the 1st successful HSCT was achieved in a patient with SCID
Lymphopoiesis in SCID patients pre/post hematopoietic stem celltransplantation
(HSCT)
Myeloid cellsMyeloid cells
Pre HSCTPre HSCT
HSCHSC
Early post HSCTEarly post HSCT
Donor
TT
NKNK
Late post HSCTLate post HSCT
TTExhaustion Exhaustion
ofofprecursorsprecursors
No donor cells in bone marrowNo donor cells in bone marrow
ImmunodeficiencImmunodeficiency !y !
Occurrence of severe chronic skin HPV disease
in SCID patients after HSCT
c, JAK3 c, JAK3 deficienciesdeficiencies
Occurrence of severe chronic skin HPV disease
in SCID patients after HSCT
• An unexpected role for An unexpected role for c/JAK-3 cytokine c/JAK-3 cytokine receptorreceptor
signaling pathways in defense of keratinocytes signaling pathways in defense of keratinocytes against papilloma virusagainst papilloma virus
New therapeutics ?New therapeutics ?
• A significant problem with no simple A significant problem with no simple solutionsolution
Principle of ex vivo gene therapy of the SCID-X1 condition
T Lymphocytes development after gene therapy of SCID-X1
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 10
20
30
40
50
60
Months after Gene Therapy
T
Lym
ph
ocy
tes/
µl
P1
P2P
7
P8
P10
P9
P4
P5
P6
*
*
**
70
High proliferation rate enables T cell diversification from a few precursors
Hematopoietic
stem cell
Myeloid cells
CLP
TransductiTransductionon
1 clone
c-dependentproliferation
TCRTCRrearrangemenrearrangemen
tsts
Multiple T cell
clones
1 integration site,
diverse TCR
As in the reversion case, oligoclonal lymphopoiesis
polyclonal repertoire
T L
ym
ph
ocyte
s/µ
l
1
10
100
1000
10000
100000
1000000
0 5 10 15 20 25 30 35 40
P4
46, XY, 6q21;13qter
• Monoclonal T cell clone
Monoclonal T cell proliferation P4
Serious adverse effect caused by retroviral insertional mutagenesis in a
protooncogene
11 22 33 44 55 66
11 22 33 44 55 66
MFG MFG cc
44,218 46,229 54,614 66,467 66,867 71,646 76,883
LMO-2 mRNA2. 3 kb
1077 1513
MFG MFG ccP5 P4
Aberrant expression ---> clonal proliferation
Enhancer activity
Clonal lymphoproliferationSynergistic effect with the c transgene
T cell clone
• 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…
Conclusion
• But… a serious adverse effect vector modifications
Vector modifications
suicide gene
LTR c
Promoter IRES
LTR
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
Under the European Science Foundation Coordinated by GIS – Institut des maladies rares
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
Partners and supporting institutions
Pharmaceutical partners
Aventis
Glaxosmithkline
Roche
Servier
Supporting institutions
Austria, Medical University Vienna Belgium, FNRS - Fonds national de
la 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 boardAssessment by a scientific advisory board
- analysis of the accuracy of the proposal
- advices
www.institutmaladiesrares.netwww.institutmaladiesrares.net
www.erditi.orgwww.erditi.org