Research on rare diseases follows a long and complex pathway (1)

1. Mendelian Genetics Gene identification Genotyping Sequencing «Variants»(same phenotype without mutation) DIAGNOSIS Validation Genotype/phenotype correlation ? PROGNOSIS Gene modifiers Pathophysiology Bioinformatics, cell imaging… Cell studiesGene expression analysis Animal models Animal transgenic facilities Phenotyping facilities (Imaging…) In vivo studies Research on rare diseases follows a long and complex pathway (1) cohortclinical expertisedatabasestechnical platforms Identification of a clinical phenotype

2. Vectorology large animals facilities in vivo Animal models Clinical trials specific methodology THERAPEUTICS Research on rare diseases follows a long and complex pathway (2) Therapeutic research Compounds libraries Screening Industrial Collaborations Targets Pathophysiology Cell and gene therapy Biological molecules Screening of molecules Class of molecules in vitro

3. PID diagnosis laboratory Dpt of Biotherapy research U550 U429 Pediatrics UIH Infectiology, immunology adults Data base center The "reference center" for primary immunodeficiencies (PID). Necker University Hospital Paris FacultédeMédecine

4. HSC T T THYMUS Myeloid compartment B SCID diseases Fatal conditions early in life X-linked or autosomal recessive inheritance 1970s

5. CD8 CD4 NK B SCID diseases 1985 Abnormal thymus in SCID SCID Control CLP HSC Myeloid compartment THYMUS

6. IL-15 NK gc deficiency CLP TCD4 IL-7 HSC TCD8 B IL-4 IL-2 IL-7 IL-9 IL-15 IL-21 gc gc gc gc IL-9R gc gc IL-2Rb IL-7Ra IL-4Ra IL-2Rb IL-21R IL-2Ra IL-15Ra X-linked SCID 1985-1995 XL-SCIDc gene

7. p Chr. 10 q Genomic deletions / Splice site mutations 10-12 100 3 5-8 1-3 5-6 5 4 9 10 11 10 Control % Survival C-ter b-Lact b-CASP RS-SCID N C RS-SCID + Arte Artemis 1 Ly NK del. 7bp M1T del. 1bp R191X G118V del. 17bp S32C del. 4bp Y199X SCIDA R81X G135E ins. 2bp H35D T gd Pro T 0 1 2 3 T CD8 Gy T CD4 Precursor Pre-B B B IgG, A, E Pro B B 1990-2001 IgM "T-B-SCID" T-B-SCID

8. v j RSS RSS 61.8 Rag-1-Rag-2 Cleavage Hairpincoding ends 4.5 v j Artemis KO 0.1 Spleen Thymus Ku70-Ku80 13.0 5.7 76.1 Hairpin opening and processing DNA-PKcs WT Excised DNA Artemis j v 8.7 XRCC4, ligase 4 0.2 0.3 KO End joining IgM CD8 1.8 B220 CD4 Generation of coding joints T-B-SCID TCR and BCR genes rearrangements during T and B cell differentiation 2001-2004

9. 1.Prevention of cell apoptosis DNA replication (purin metabolism)ADA 2.gc cytokine-dependent signalsgc, JAK-3, IL7Ra 4.Pre TCR/TCR signalling)CD45, CD3, 3.V(D)J recombinationRag-1/-2, Artemis SCID diseases 2005 NK CD8 CLP HSC CD4 THYMUS Myeloid compartment B More diseases to be described…

10. Molecular analysis of SCID conditions, what for ? Basic science: lymphocyte development Molecular diagnosis/genetic counseling Specific therapies: ADA enzyme substitution, gene therapy 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…

11. JAK-3 inhibitor GAS Schematic representation of a major signaling pathway activated by IL-2

12. Description of multiple SCID variants Three examples: Omenn's syndrome  Hypomorphic mutations of Artemis  Revertant in XL-SCID

13. 1 1043 K86Fs. Y333• G720S R1010H R511H L732F K1029E T173Fs. R404W R404Q D832N S875Fs. Hypomorphic mutations of Rag-1 Patient Control Oligoclonal T cell expansion Omenn’s Syndrome: hypomorphic mutations in Rag-1/2emergence of autoreactive T cell clones CD3 staining skin gut Defective central T cell tolerance

14. b-Lact C-ter b-CASP “Catalytic domain” “Regulatory domain” Occurrence of B cell lymphomas CD20 Ki67 Hypomorphic mutations ofArtemisassociated with B cell lymphomas Hypomorphic mutationsPhenotype ? T432 fs D451 fs Role of Artemis as a genome caretaker

15. An atypical case of XL-SCID gc deficiency NK NK Reverse mutation TCD4 TCD4 gc gc HSC TCD8 TCD8 gc gc gc B B T cell counts = 50 % of controlmemory phenotype Repertoire diversity generated from one precursor (revertant cell) ? Absence of T cellsAbsence of NK cells

16. 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 !

17. T cells/µl 3,000 HLA id. Homeostatic expansion of donor mature T cells 2,000 Haplo id. New T cell ontogeny 1,000 months 0 1 2 3 6 12 4 Persisting severe immunodeficiency 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 HLA haplo id. parent Survival % Months European registry - 464 pts

18. Early post HSCT Late post HSCT NK Donor T T Immunodeficiency ! Exhaustion ofprecursors No donor cells in bone marrow Lymphopoiesis in SCID patients pre/post hematopoietic stem celltransplantation (HSCT) Pre HSCT HSC Myeloid cells

19. Occurrence of severe chronic skin HPV disease in SCID patients after HSCT c, JAK3 deficiencies

20. Occurrence of severe chronic skin HPV disease in SCID patients after HSCT • A significant problem with no simple solution • An unexpected role for c/JAK-3 cytokine receptor signaling pathways in defense of keratinocytes against papilloma virus •  New therapeutics ?

21. Principle of ex vivo gene therapy of the SCID-X1 condition

22. 9000 8000 * P4 7000 6000 P8 5000 * T Lymphocytes/µl P9 4000 3000 P2 P7 * 2000 P5 P6 1000 P1 P10 0 0 10 20 30 40 50 60 70 Months after Gene Therapy T Lymphocytes development after gene therapy of SCID-X1

23. Transduction Multiple T cell clones1 integration site, diverse TCR 1 clone CLP c-dependentproliferation TCRrearrangements Myeloid cells Hematopoieticstem cell High proliferation rate enables T cell diversification from a few precursors As in the reversion case, oligoclonal lymphopoiesis  polyclonal repertoire

24. 1000000 100000 10000 T Lymphocytes/µl 1000 100 P4 10 1 40 0 5 10 15 20 25 30 35 Monoclonal  T cell proliferation P4 46, XY, 6q21;13qter • Monoclonal   T cell clone

25. Serious adverse effect caused by retroviral insertional mutagenesis in a protooncogene 44,218 46,229 54,614 66,467 66,867 71,646 76,883 MFG gc MFG gc 1 2 3 4 5 6 P5 P4 Enhancer activity Aberrant expression ---> clonal proliferation LMO-2 mRNA 2. 3 kb 1 2 3 4 5 6 1077 1513 T cell clone Clonal lymphoproliferation Synergistic effect with the c transgene

26. 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

27. Vector modifications  gc LTR LTR Promoter suicide gene IRES  U3 Self inactivated LTRloss of the enhancer effect

28. 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

29. 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

30. 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

31. 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 Partners and supporting institutions

32. 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