Research on rare diseases follows a long and complex pathway (1) Identification of a clinical phenotype Mendelian Genetics Genotyping Sequencing Gene identification.

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