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 Lentivirus Lentivirus
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
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
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
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
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
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
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
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
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+
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
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
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
Outcome of ADA-SCID GT T cell NK cell B cell Erythrocyte Platelet Granulocyte Monocyte
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
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
Current Treatment Filipovich, Blood 2001; Kobayashi, 2006; Ozsahin, Blood 2008
LENTIVIRAL VECTORS WASP CMV GA RRE cPPT was PRE • HIV derived, self-inactivating system • Safer integration profile • Physiological promoter • Improved GT efficiency into HSC
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
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 -/-
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)
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
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
Gene/cell therapy for rare diseases “Old” therapy approaches Very rare population! “Personalised” therapy Single curative injection
• 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
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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