Inserm. Gene Therapy of Human beta-thalassemias. Emmanuel Payen Chiang Mai : 2013, March 31

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1 Inserm Gene Therapy of Human beta-thalassemias Emmanuel Payen Chiang Mai : 213, March 31 Institut des Maladies Emergentes et des Thérapies Innovantes (imeti) Service des Thérapies Innovantes (STI) Laboratoire de Thérapie Génique et Cellulaire (LTGC) CEA, Fontenay aux Roses

2 Rational for gene therapy in patients with β-thalassemia Hematopoietic stem cell transplantation (HSCT) offers the only hope of cure In children, the balance of risks and benefits favors allogeneic HSCT over medical therapy for those who have an HLA-matched sibling but: - Only 25% patients have a non-affected HLA matched sibling - In adult patients, allogeneic transplant is characterized by high toxicity due to advanced disease (transplant related mortality 3% and thalassemia free survival 7% ; Gaziev Ann N Y Acad Sci 25; La Nasa BMT 25); uncertain risk/benefit balance Gene therapy with autologous HSCT would bea solution for those who do not have a compatible donor and is expected to be associated with lower transplant related mortality and morbidity versus allogeneic SCT - Conditioning with only busulfan, no cyclosphosphamide or other immunosuppressants - Avoid risks of graft rejection, GVHD

3 The many mutations causing β-thalassemia makes gene addition the preferred approach

4 HPV569: Lentiviral vector with a β-globin β A-T87Q gene and core insulators 2xcHS4 Insulator cores 2xcHS4 Insulator cores human β-globin gene Ψ + cppt/flap RRE III 3 enhancer II 266bp 644bp 845bp 1153bp I βp HS2 HS3 HS4 ppt SIN + Insulator β A-T87Q SIN + Insulator Why β A-T87 marking / mutation? In-vivo biomarker for transgene detection Provides anti-sickle properties (Science 21) Absrob bance 22 nm β A-T87Q β E β A α γ G γ A BERK MOCK BERK β A-T87Q

5 LG1: Phase I / II Clinical Trial (France) A phase I/II open label study with anticipated benefit evaluating genetic therapy of the β-hemoglobinopathies h l (sickle cell anemia and β-thalassemia l i major) by transplantation of autologous CD34+ stem cells modified ex-vivo with a lentiviral β A-T87Q globin (Lentiglobin TM ) vector (Eliane Gluckman, M Cavazzana-Calvo PI; Philippe Leboulch SD) 1 patients (5-35 yo) - thalassemia major - sickle cell anemia Started 27

6 Overview of the clinical protocol Bone Marrow or PB Harvest (2x1 8 unsorted BM cells/kg kept for rescue) CD34+ cells Busulfex Vector + Cytokines Maximize % Transduced HSCs Testing and Release While Frozen Bone Marrow Conditioning Maximize Myeloablation Without Immunosuppression IV Infusion Transduced Cells ( 4x1 6 CD34 + /Kg) (Spontaneous Homing)

7 Nomenclature and transplantation schedule of patients Patient 12 : Injection of backup cells Patient 13 : Transplantation > 5 years ago Patient 14 : Transplantation > 18 months ago

8 Pre-transplant clinical history of patient 13 with severe β E /β -thalassemia Then 18 year old male with β E /β -thalassemia and no HPFH or α mutation Transfusion dependent since age 3 (2-3 RBC packs every month, > 225 ml RBCs /kg/year for Hb > 1 g/dl) Spontaneous Hb levels as low as 4.5 g/dl Major hepato-splenomegaly (splenectomy at age 6) and growth retardation Failure of Hydroxyurea therapy (between ages 5 and 17) Desferoxamine (5 days/week) since age 8, and oral Exjade since age 18. No liver fibrosis. Moderate iron overload by liver MRI (561 μmol/g). No related, genoidentical HLA-matched donor. Match strict inclusion and exclusion criteria Transplantation > 5 years ago, at age 18 on June 7, 27 Uneventfull - Duration aplasia = 35days

9 Vector copy numbers per circulating cell (>5 years post-transplantation) Ve ector cop py / cell CD15 (neutrophils) CD14 (monocytes) CD19 (B lymphocytes) Whole blood CD3+ (T lymphocytes) CD45-CD71+ (erythroblasts) Months post-transplantation

10 Percentages of vector bearing cells in bone marrow and blood cells BONE MARROW (36 months) All nucleated cells 21. % CD % Erythroblastes (glycoa+ CD71+) 34.6 % Myeloid progenitors (CFU-GM) 11.4 % Erythroid progenitors (BFU-e) 22.6 % BLOOD (35 months) All nucleated cells 9. % Granulocytes-Monocytes (CD15+) 17.9 % B Lymphocytes (CD19+) 9.2 % T Lymphocytes (CD3+) 2.3 % Erythroblastes (CD45- CD71+) 1.7 % 2% g (%) ctor bearing blood cells Ve whole Whole blood Months post-transplantation Memory T cells, not exposed Dyserythropoiesis

11 Conversion to transfusion independence for > 4 years (13) Transplantation Last transfusion Hemoglo obin (g/d dl) Phlebotomies >4 y without transfusion 8-9 g / dl Months after transplantation Transfusion Bleeding

12 Conversion to transfusion independence (II) Last transfusion > 5 years ago 12 Phlebotomies (2 ml each) β A-T87Q α β E γ G γ A He emoglobin (g/dl) Total Hb (produced) g/dl vector-encoded βa T87Q -globin HbA T87Q HbF HbE Months post-transplantation HbA (transfused)

13 Improved Erythropoiesis and RBC Survival roblasts (x x1 3 /µl) Eryth Decreased circulating erythroblasts t Increased RBC lifespan Months post-transplantation / µl) RBC (x Retic (x1 6 / µl) Months post-transplantation Decreased Iron Overload Phlebotomies Ferritin (µg g/l) Months post-transplantation

14 Integration site (IS) analysis by LM-PCR and DNA pyrosequencing (whole nucleated blood cells and purified sub-populations) 1% 8 6 Low total number of different IS (< 3) In actively transcribed regions, similar to generic HIV vector 24 IS both myeloid and lymphoid % among transduced cells HMGA2 HMGA2 FBXL11 TBC1D5 PILRB MKLN1 IRAK1 ZZEF1 RFX3 NUP98 4 ATXN1 EPB41L2 2 EIF1 PHF16 SAE GNA12 Months post-transplantation (whole nucleated blood cells) Relati e dominance of IS at the HMGA2 loc s Relative dominance of IS at the HMGA2 locus (dominance relative to other IS, but > 8 % cells remain untransduced) POLA2

15 Aberrant splicing of the third intron and polyadenylation within the globin lentiviral vector ATG Globin-LV stop Let-7 mirnas E1 E2 E3 E4 E5 Intron 3 (~ 113 kb) Deletion of 1 copy insulator core Sequencing of the main HMGA2 transcript: Aberrant splicing within the vector insulator + polyadenylation ERYTHROID-SPECIFIC EXPRESSION Relative expression Months post BMT

16 Hematopoietic homeostasis is maintained Normal blood and bone marrow cytology Normal cytoflurometry analysis Normal karyotype and high resolution CGH-array chromosomal analysis No Trisomy 8 with specific probe Lack of cytokine-independence in CFU-C assays Normal LTC-IC counts No preferred engraftment in NSG mice (coll. C. Eaves) No abnormality detected for MPL, JAK2 and TET2 Natural cases of Paroxysmal Noctural Hemoglobinuria (PNH) with complete hematopoietic dominance of HMGA2 dysregulated clone followed without malignancy for ~ 2 years Decrease of the clone relative dominance

17 The percentage of partially dominant HMGA2 site is decreasing over time in whole nucleated blood cells (qpcr 5 years post-transplant) 18 copies HMGA2 modified whole bloo od cells (% %) Ge enetically LentiGlobin vector LentiGlobin vector at HMGA

18 qpcr based kinetics of the four most common IS up to 5 years post-transplantation transplantation.14 Vector cop py numbe er copies HMGA2 RFX3 FBXL11 ZZEF Months post-transplantation transplantation 4 years ZZEF1 = 1/3 HMGA2 5 years ZZEF1 = HMGA2

19 HMGA2 IS in other gene therapy trials Evidence for hot spot and homeostatic control of cell expansion HMGA2 in X-SCID trial (γ-rv vector) > 15 cluster IS in HMGA2 (aggregates of patients data): - 12 in HMGA2 Intron 3-11 in same orientation - Increase abundance with time and then stabilize - 2(at least) with truncated RNA by aberrant splicing Intron 3 into vector HMGA2 in ALD trial (LV vector) 1 IS in HMGA2 Intron 3 in patient P1: - only in B lymphocytes and 1 time-point (9 months) Wiskot Aldrich trial (γ-rv vector) MGMT glioblastoma trial (γ-rv vector) Lentiviral vectors have preferential target regions in the absence of overt tumorigenesis and selection Tubsuwan A., Abed S.,. Maouche-Chretien L, Cell Stem cell 213

20 Second β E /β -thalassemia (major) gene therapy patient transplanted on November 24, year old woman, β E /β Thal Major Transfusion dependent d since her 2 nd month of life. No HLA matched sibling donor The transplantation was uneventful. Engrafted neutrophils by day 2 and had delayed ed platelet reconstitution tion (no related complications) 4.3 X 1 6 / kg total CD34+ cells infused Low transduction efficiency The patient has returned to full time work With ih24 months follow up, substantially lower VCN and expression of βa T87Q compared to prior patient. Transfusion frequency is being compared to pretransplant data One HMGA2 site in the erythroid compartment, 6 months post transplantation

21 How to improve the risk / benefit balance? Reduce the risk for insertional mutagenesis target vectors to safe harbours homologous recombination Insert a suicide gene Increase the number of corrected stem cells Improve transduction efficacy Select for transduced cells

22 Limited transduction efficiency (1-2% in long term stem cells) Cavazzana-Calvo M., Payen E.,. Leboulch P. Nature 21 β-thal (Patient 13) ) Vector bearing whole blo od cells (%) Months post-transplantation vect tor bearing ells (%) CD15 CD14 CD19 ccd Months post-transplantation CD45-CD71+ Cartier N., Hacein-Bey-Abina S.,.., Aubourg P. Science 29 ALD (P1+P2) WB BC expressin ng ALD protein (% %) Months post-transplantation WBC express sing ALD protein (%) Months post-transplantation

23 Ambivalent effect of the chs4 chromatin insulators + Higher probability for transgene expression - Reduced vector titer - Reduced primary cell transduction efficacy - Minimal overall efficacy In vitro Insertional Mutagenesis Activity DL γ-retroviral vector SF-GFP LentiViral vectors SF-GFP LCR-βp-GFP + INS LCR-βp-GFP - INS 1.7X Mock vector βp-gfp - INS In vitro genotoxic potential (cell replating frequency / vector copy) Arumugam; Mol Ther 29

24 Optimization of vector backbone : increased titer Remove the chromatin insulator sequences and change the viral promoter HPV569 HPV524 BB35 HIV R HIV R CMV R cppt/flap RRE cppt/flap RRE cppt/flap RRE βe βe βe β-globin β A-T87Q β-globin β A-T87Q β-globin β A-T87Q βp βp βp LCR(2.7kb) HS2 HS3 HS4 LCR(2.7kb) HS2 HS3 HS4 LCR(2.7kb) HS2 HS3 HS4 2x25 chs4 SINU3 SINU3 pa R pa R pa R 5x1 7 TU/mL 7x1 7 TU/mL 1x1 7 TU/mL Increa ased tite er

25 Increased titer and improved purification = Increased potency HPV569: 1 x 1 8 TU/mL ; particle (ng p24 ) /infective (TU) 1 BB35: 4 x 1 8 TU/mL ; particle (ng p24 ) / infective (TU) 3 CD34 β A /β A, MOI=12.5 to 5 1,2 CFCs Co opy per ce ell 1,,8 3X,6,4,2, MOI HPV BB35 Co opy per ce ell 1,2 1 1,,8,6,4,2, CD34 β s /β E, MOI=25 2X 1,2 1 1, 3X,8,6,4,2, CFCs Co opy per ce ell LTC-ICs β AT87Q α β AT87Q α β E β S β S β E γ G γ A γg γ A γ A

26 In vivo safety profile for the New drug product U3 cppt/flap R RRE βe β A-T87Q β-globin βp LCR 2x25 chs4 pa R CMV cppt/flap R RRE βe β A-T87Q β-globin βp LCR SIN R pa LentiGlobin TM (HPV569) LentiGlobin TM (BB35) Donor cells = β Thal mice (CD45.2) Primary recipients 58 β Thal mice (CD45.2) 4 months BM cells Necropsy, Histopathology, hematopoietic homeostasis, vector insertion sites Secondary recipients 18 normal C57BL/6 mice (CD45.1) 6 months Necropsy Histopathology Necropsy, Histopathology, hematopoietic homeostasis, vector insertion sites

27 Comparable therapeutic efficacy Male bone marrow Female bone marrow Mean VCN Mean VC CN HPV569 BB35 Mock HPV569 BB35 Mock Therapeutic Hemoglobin Total Hemoglobin Reticulocytosis Percen ntage (%) Hb (g/dl) Reticulo ocytes (%) Males Females Males Females Males Females

28 Similar and expected insertion site distribution Primary recipients Secondary recipients te (%) Integ gration si In gene In gene +/-1kb In gene In gene +/-1kb ion site (% %) Integrat 8 Distribution in genes and upstream HPV569 BB35 In Silico upstream In gene

29 Similar frequency of tumor development among the 18 secondary mice Males Females Vector treatment HPV569 BB35 MOCK HPV569 BB35 MOCK Lymphomas or leukemias / number of animals 3/21 3/18 /9 4/23 3/18 2/19 No statistically significant difference in proportions of mice with lymphomas or leukemia (p.4) No detection of the vector in tumor cells

30 Supporting evidences for take off (Leboulch, Nature 213) Science Vol November 6, 29 Science Vol August 213 Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy Cartier N, Hacein-Bey Abina S,. Aubourg P ( 2% Tdx blood cells) Lentiviral Hematopoietic stem cell gene therapy benefits metachromatic Leukodystrophy Biffi A, Montini E,. Naldini L (>6% Tdx blood cells) Nature Vol 467 September 16, 21 Transfusion independence and HMGA2 activation after gene therapy of human β-thalassaemia Cavazanna Calvo M, Payen E,. Leboulch P ( 2% Tdx blood cells) Science Vol August 213 Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Aiuti A, Biasco L. Naldini L (25-5% Tdx blood cells) CMV R cppt/flap RRE βe β A-T87Q -globin βp LCR SIN R pa Much better transduction efficiency with the new LentiGlobin vector Higher titer + higher purity

31 HB25: Phase I / II Clinical Trial (France) (follows LG1) Continuation study with a revised clinical protocol based on the use of a modified LentiGlobin vector (BB35) (M Cavazzana-Calvo PI; Philippe Leboulch SD) 7 patients (5-35 yo) - thalassemia major - sickle cell anemia Clinical trial authorization (CTA) active ; Clinical trial authorization (CTA) active ; study initiated : patient being enrolled

32 HGB24: Phase I / II Clinical Trial (USA) A phase I/II open label study evaluating efficacy and safety of gene therapy in subjects with β-thalassemia l i major by transplantation t ti of autologous CD34+ stem cells modified ex-vivo with a lentiviral β A-T87Q globin (Lentiglobin TM BB35 Drug product) (PI: Mark Walters) 15 patients t (5-35 yo) - thalassemia major Investigational new drug application (IND) active; Investigational new drug application (IND) active; Multicenter trial approved by RAC and FDA: patients being enrolled

33 Lentiviral vectors for gene therapy of β-thalassemia / sickle cell disease : Paris, Cambridge MA : Philippe Leboulch, bluebird bio LentiGlobin TM U3 R cppt/flap β A-T87Q LCR(2.7kb) chs4 2x25 RRE e β β-globin p β HS2 HS3 HS4 pa R New York: Michel Sadelain TNS CMV R RRE βe β A -globin βp LCR(3.2kb) HS2 HS3 HS4 SIN R WPRE pa Milan: Giuliana Ferrari Globe Vector RSV R cppt/flap RRE βe β A -globin βp LCR(2.7kb) HS2 HS3 SIN R U5 Cincinnati: Punam Malik sbg 65 Vector RSV R RRE βe β A -globin βp LCR(3.1kb) HS2 HS3 HS4 65 chs4 R U5 Memphis: Derek Persons V5m3-4 Vector CMV R cppt/flap RRE βe γ-globin βp LCR(3.1kb) HS2 HS3 HS4 4 chs4 R U5

34 Clinical trial Acknowledgements - Inserm U962 / CEA-iMETI / University Paris 11 / bluebird bio France Olivier Negre, Yves Beuzard, Floriane Fusil, Beatrix Gillet-Legrand, Céline Courné, Anaïs Paulard, Emmanuel Payen, Philippe Leboulch - bluebird bio (Cambridge, MA) Gabor Veres, Maria Denaro, Robert Kutner, John Piercey, Rob Ross, Dave Davidson, Mitchell Finer - Necker Hospital Salima Hacein-Bey Abina, Laure Cacavelli, Felipe Suarez, Jean-Antoine Ribeil, Olivier Hermine, Marina Cavazanna Calvo - Other hospitals (St-Louis, Mondor, CHIC, Tenon) Eliane Gluckman, Françoise Bernaudin, Frédéric Galacteros, Gérard Socié, Jean Soulier, Robert Girot, Nabil Kabbara - University of Pensylvannia, school of Medicine, Philadelphia Gary Wang, Troy Brady, Frederic Bushman Vector, transduction, cell culture - Inserm U962 / CEA-iMETI / University Paris 11 / bluebird bio US / bluebird bio France Olivier Negre, Robert Kutner, Byoung Ryu, Garrett Heffner, Suparerk Borwornpinyo, Yves Beuzard, Maria Denaro, Beatrix Gillet- Legrand, Céline Courné, Anaïs Paulard, Emmanuel Payen, Philippe Leboulch - Indiana Vector Production Facility, Indianapolis, IN Scott Cross, Chris Ballas, Ken Cornetta Thalassemic IPS cells characterization and derivation - Inserm U962 / CEA-iMETI / University Paris 11 Leila Maouche-Chrétien, Alisa Tubsuwan, Soumeya Abed, Philippe Leboulch - Mahidol University, it Bangkok k Alisa Tubsuwan, Suthat Fucharoen

35 Hematopoietic homeostasis is maintained in mouse overexpressing HMGA2 EGFP vector TrHMGA2 + EGFP vector FL HMGA2 + EGFP vector EG GFP (%) In vitro M Tr FL EGF FP+ (%) Myelo Months (x 1-9 / L) count 6 * Myelo 5 * 4 * * Months (%) EGFP+ 1 WBC Months -12 / L) count (x WBC * Months (%) EGFP+ 1 LB Months -9 / L) count (x 1 8 LB * Months * EGFP+ (%) 1 8 RBC Months 12 cou unt (x 1-12 / L) * 12 RBC * 1 * Months EGFP+ (%) 1 LT Months cou unt (x 1-9 / L) LT Months