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ORPHAN DRUGS Alessandro Aiuti UNIVERSITA DI ROMA TOR VERGATA iget - PowerPoint PPT Presentation

GENE THERAPY-BASED ORPHAN DRUGS Alessandro Aiuti UNIVERSITA DI ROMA TOR VERGATA iget TELETHON INSTITUTE FOR GENE THERAPY Gene therapy strategies In vivo approaches Ex vivo approaches Adenovirus Retrovirus AAV Adenovirus DNA/Liposomes


  1. GENE THERAPY-BASED ORPHAN DRUGS Alessandro Aiuti UNIVERSITA’ DI ROMA TOR VERGATA iget TELETHON INSTITUTE FOR GENE THERAPY

  2. Gene therapy strategies In vivo approaches Ex vivo approaches Adenovirus Retrovirus AAV Adenovirus DNA/Liposomes Lentivirus Lentivirus

  3. Gene therapy-based EU designated Orphan Drugs Ex vivo In vivo Duchenne Muscular Distrophy Alpha-Sarcoglycanopathy Muscle SCID-X1 Gamma-Sarcoglycanopathy Hematopoietic ADA-SCID System Wiskott-Aldrich Syndrome Glycogen storage dis. type II Multiple Beta-thalassemia (Pompe's disease) organs Retinitis pigmentosa CNS & Leber’s amaurosis Metachromatic Eye PNS Stargadt’s disease Leukodystrophy Epidermolysis bullosa Skin

  4. Primary immunodeficiencies INNATE IMMUNITY ADAPTIVE IMMUNITY ADA-SCID ADA-SCID WISKOTT-ALDRICH SYNDROME • Alterations in development and/or functions of adaptive/innate immunity • Higher susceptibility to infections • Failure to thrive • Increase risk of autoimmunity and cancer

  5. HSC gene therapy for primary immunodeficiencies Medicinal Product Gene transfer Autologous vector gene modifed HSC Starting material -Autologous procedure ( No rejection/ GVHD) - Reduced toxicity - Selective advantage for gene corrected cells - Data on safety and efficacy from preclincial studies and pilot studies

  6. Adenosine Deaminase-deficient SCID Autosomal recessive dAdo, Ado Adenosine 1:375,000-1,660,000 dAXP deaminase • Bone and growth abnormalities • T, B, NK, lymphopenia • Organ toxicity (lung, liver) • Severe recurrent infections • Neurological and behavioral alterations • Autoimmunity TREATMENT OPTIONS  Bone Marrow Transplantation  Enzyme Replacement Therapy (PEG-ADA)  HSC Gene Therapy

  7. RATIONALE FOR GENE THERAPY  Scientific rationale • The ADA gene is constitutively and ubiquitously expressed • Gene-corrected lymphocytes have an advantage over ADA-deficient cells. • 10% of normal ADA expression may be sufficient  Unmet medical need • 90% of children lack an histocompatible donor in the family • High risk of bone marrow transplant from alternative donors (30-65% survival) • Treatment with bovine enzyme (PEG-ADA) (80% survival) not a definitive cure, not always effective, very expensive

  8. Gene transfer protocol into autologous bone marrow CD34 + cells No PEG-ADA BM Harvest Busulfan 2 mg/Kg/day x 2 (days -3, -2) Day -4: Purification Day 0: Infusion of BM CD34 + cells SV Neo ADA MLV LTR Days -3 to -1: Day-4: Prestimulation 3 cycles of transduction (TPO, FLT3-ligand, on retronectin + cytokines SCF, IL-3) A. Aiuti, MG Roncarolo, C. Bordignon, 2002

  9. Long-term engraftment of gene corrected cells CD15+ granulocytes CD3+ T cells 100 100 % of vector of positive cells % of vector of positive cells 10 10 1 1 0.1 0.1 <0.01 <0.01 0 1 2 3 4 5 6 7 0 1 2 3 4 5 6 7 Years after GT Years after GT CD19+ B cells CD34+ progenitors 100 100 % of vector of positive cells % of vector of positive cells 10 10 1 1 0.1 0.1 <0.01 <0.01 0 1 2 3 4 5 6 7 0 1 2 3 4 5 Years after GT Years after GT A. Aiuti et al. NEJM, 2009

  10. POLYCLONAL VECTOR INTEGRATIONS and REPERTOIRE Diversity of integrations in T lymphocytes TCR Vbeta Repertoire Proportion
of
different
clones
 20,0 4 yrs after infusion Pt1 12 ND (n=46) Pt4 Pt7 12 12 10 15,0 10 10 8 8 8 6 6 6 10,0 4 4 4 % TCRVbeta in CD3 + 2 2 2 lymphocyte subset 0 0 0 5,0 12 12 12 0,0 Pt2 Pt5 Pt9 10 10 10 0
 20
 40
 60
 80
 100
 120
 140
 8 8 8 6 6 6 4 4 4 2 2 2 12 No in vivo skewing 0 0 0 10 8 12 12 Pt3 Pt6 Pt10 10 6 10 8 4 8 6 2 6 4 0 4 2 2 0 0 Aiuti et al., JCI 2007 and unpublished results

  11. Immune reconstitution after GT Thymus 3 yrs post-GT 1500 Cells/ µ l 1000 500 0 0 +3 years +1 year +2 years +3 years (n = 8) (n = 6) CD3+ CD4+ Median (n=9) CD8+

  12. T- cell reconstitution after gene therapy Recovery of thymic functions Recent thymic T-cell functions emigrants (anti-CD3) anti-CD3 mAb 10000 * 300000 200000 Copies / 100 ng of DNA 100000 50000 1000 cpm 5000 100 10 100 pre-GT 6 months 1 year 2 years 3 years Healthy controls n=9 n=9 n=9 n=7 n=5 n=114 PRE-GT 1y FU 2y FU >3y FU BMT HC Aiuti et al., NEJM, 2009

  13. Immune response and protection from infections • IVIg discontinued in 6 pts with proven antibody response (TT, DT, Pertussis, Haemophilus, Pneumo) 2 Pts ongoing IVIg discontinuation • MMR vaccine in 1 pt led to protective antibodies Rate of severe infections • Four patients experienced varicella 15 without complications N. events/10 person-months 10 5 0 Pre GT Post GT 20 infections/ 215.4 person-month 4 infections/ 394.5 person-month

  14. Systemic detoxification and growth improvement dAXP metabolites 500 400 nmoles/ml 300 200 * 100 0 0 1 2 3 4 5 6 7 Years after GT

  15. Outcome of ADA-SCID GT T cell NK cell B cell Erythrocyte Platelet Granulocyte Monocyte

  16. Clinical translation in ADA-SCID FDA EMEA ODD ODD 2009 Towards 2005 1991 2000 2008 2002 registration Pilot studies Phase I/II First attempts Enrolment closed Nonclinical EMEA protocol assistance

  17. Wiskott-Aldrich Syndrome (WAS) Plasma CELLULAR DEFECTS GTP membrane PIP2 Cdc42 HSC migration Nck T cells migration, immune syn. Tec SH3 B cells migration, Ig production Grb2 WIP Platelets reduced size / number PH-EVH1 GBD Dendritic cells migration Actin monomer Pro Macrophages adhesion, antigen uptake NK cells cytotoxic activity Active WASP A C V V Actin polymerization Autoinhibited WASP Arp2/3 X-linked, 1,250.000 newborn male Cytoplasm  Eczema  Bleeding Life expectancy: 15 years in severe forms  Infections (WAS-negative)  Autoimmunity  Tumors

  18. Current Treatment Filipovich, Blood 2001; Kobayashi, 2006; Ozsahin, Blood 2008

  19. LENTIVIRAL VECTORS WASP CMV GA RRE cPPT was PRE • HIV derived, self-inactivating system • Safer integration profile • Physiological promoter • Improved GT efficiency into HSC

  20. SAFETY AND EFFICACY OF WAS GT IN WAS KO MICE Transplantation
i.v.
 2.5x10 5
 ‐ 
 10 6 
lin ‐ 
BM
cells 
 BM
harvest
 12
hrs
 700
rads
(sublethal) 
 WAS‐/‐ 
donor
 (male/CD45.2)
 LV
transduction
 WAS‐/‐ 
recipient
 w1.6W_WPREmut 
 (female/CD45.1) h1,6WASP 
 RSV hWASP
 WPRE
 GA RRE cPPT Engraftment and selective advantage Low MOI High MOI * * * * * 100 100 98 mice followed for 4-16 months cells cells 75 * 75 No long-term toxicity + * + % WASP % WASP 50 50 No vector derived tumors 25 25 0 0 No increase in tumor incidence + CD11b + B220 + + + + CD11b + B220 + + + CD45 CD8 CD4 CD45 CD8 CD4 BM Spleen BM Spleen

  21. FUNCTIONAL CORRECTION OF T-CELLS AND B CELLS T-cell functions Ab TITER d=7 Pneumo23 vaccine challenge IL-2 350 10000 ** TP23 WT * ** ** 300 TP23 UT * 1000 (ng/ml) TP23 GT 250 100 200 10 150 1 anti-CD3 100 2 µ g/ml 50 PROLIFERATION 10000 ** 0 ** ** TP23 WT TP23 UT TP23 GT 1000 SI 100 10 1 wt w1.6W high MOI Lin - Was -/- F. Marangoni, A. Villa, M. Bosticardo w1.6W low MOI Was -/-

  22. SUMMARY OF TOXICITY AND SAFETY STUDIES (CD34+ cells) In vitro growth Biodistribution Colony assay (CFC,LTC-IC) Vector shedding Vector shedding Germline transmission Vector integrations 300+250
rads 
 Rag2 ‐/‐
 γ c ‐/‐ 
(neonates) 


  23. The path to clinical trial in WAS • Lack of toxicity • Safety and efficacy in the animal model • Selective advantage for gene corrected cells • Efficient gene transfer in human CD34+ stem cells 2002 2006 2008 2010 2004 Vector & Preclinical Manufacturing Validation Phase I/II Proof of concept studies and quality trial

  24. Design: non-randomized, open label, single center Population: 6 patients -Severe WAS mutation or WAS clinical phenotype -No HLA-identical sibling -No HLA-matched UR BM or UCB donor (Pts <5 yrs) Follow up: 3 years, then long-term safety protocol Study objective: Evaluate the safety, biological activity and efficacy of GT Financial sponsor: Fondazione Telethon PI: A Aiuti, MG Roncarolo, Co-PI: Fabio Ciceri Authorized by Ethical Committee (12/09) and iget National authority (ISS) (03/10) TELETHON INSTITUTE FOR GENE THERAPY

  25. Gene/cell therapy for rare diseases “Old” therapy approaches Very rare population! “Personalised” therapy Single curative injection

  26. • Often very rare populations • Long-term safety • Mainly academic-driven, high costs • Limited interest for pharma company ’ s investment • Manufacturing and standardization • Rapidly evolving scientific field and regulation

  27. Partners required for clinical development of gene therapy-based ODD Industrial Patients’ Funding agencies partner organization European Investigators National Community (PRECLINICAL AND CLINICAL) Health system Manufacturing Manufacturing (Biotech or Academic) (Biotech) EC Regulatory Agencies Regulatory Agencies (National level)

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