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Five year outcome of lentiviral gene therapy for human beta-thalassemia, lessons and prospects

Five year outcome of lentiviral gene therapy for human beta-thalassemia, lessons and prospects. Cyprus TIF 2012 - P. Leboulch.

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Five year outcome of lentiviral gene therapy for human beta-thalassemia, lessons and prospects

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  1. Five year outcome of lentiviral gene therapy for human beta-thalassemia, lessons and prospects Cyprus TIF 2012 - P. Leboulch

  2. The only curative treatment for the β-thalassemia currently is allogeneic hematopoietic transplantation, although fewer than 25% patients have an HLA-matched related donor, and those who do still face the risks of engraftment failure and Graft-Versus-Host Disease (GVHD) Ex vivo gene therapy by transfer of a therapeutic β-globin gene into the patient’s own hematopoietic stem cells (HSCs) would alleviate both donor shortage and GVHD. It is however especially challenging and a paradigm of gene therapy with no intrinsic in vivo advantage for corrected HSCs.

  3. bA-T87Q SIN + Insulator (Δ 1 core in ≈ 50%) SIN + Insulator (Δ 1 core in ≈ 50%) 2 x cHS4 Insulator cores 2 x cHS4 Insulator cores Lentiviral vector design with marked β-globin gene human -globin gene cPPT/FLAP ppt 266bp 644bp 845bp 1153bp  III II I HS2 HS3 HS4 p 3’ enhancer RRE • Why bA-T87marking / mutation? • Provides anti-sickling properties (Leboulch and coll. Science 2001) • In-vivo biomarker for detection therapeutic globin by HPLC

  4. Maximize % Transduced HSCs Vector + Cytokines CD34+ cells Testing and Release While Frozen Busulfex Bone Marrow Conditioning IV Infusion Transduced Cells (≈ 4x106 CD34+/Kg) (Spontaneous Homing) Maximize Myeloablation Without Immunosuppression Overview of the clinical protocol Bone Marrow or PB Harvest (2x108 unsorted BM cells/Kg kept for rescue)

  5. Phase I/II clinical trial: Initial focus on severe E/0-thalassemia High worldwide prevalence In Thailand alone, ~ 3,000 new cases born each year. ~ 4% of the 350 million Southern China population carry a E- or a 0- allele. High prevalence among US immigrants. Severe in ~ 50% cases (similar to 0 Thalassemia major) Transfusion dependency, iron chelation therapy and often splenectomy. Candidates for allogenic transplant when HLA-matched sibling donor. Narrow differential Hb levels between severe and well tolerated cases Mean spontaneous Hb levels separated by > 2 g/dl.

  6. Pre-transplant clinical history of the first gene therapy patient without injection of backup cells 18 year old male with severe E/0-thalassemia (major) and no HPFH or α mutation. Transfusion dependent since age 3 (> 225 ml RBCs /kg/year for Hb > 10 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 (although nausea). No liver fibrosis. Moderate iron overload by liver MRI (561 mol/g). Only child. No related, genoidentical HLA-matched donor. Match strict inclusion and exclusion criteria. Transplantation 5 years ago at age 19 on June 7, 2007

  7. Vector copy numbers per circulating cell subsets (up to 5 years post-transplantation) 0.20 0.18 Whole blood 0.16 0.14 CD15 (neutrophils) CD19 (lymphocytes B) 0.12 Vector copy / cell CD45-CD71+ (erythroblasts) 0.10 CD3+ (lymphocytes T) 0.08 CD14 (monocytes) 0.06 0.04 0.02 0.00 0 10 20 30 40 50 60 Months post-transplantation

  8. Conversion to transfusion independence for 4.4 years (5.4 years post-transplantation) Transfused Blood Published Sept 2010 Lentiglobin Treatment Transplantation on June 7, 2007 Last RBC transfusion on June 6, 2008

  9. Conversion to transfusion independence (II) Last transfusion > 4.4 years ago Phlebotomies (200 ml each) 12 10 Hb totale 8 ≈ 3.5 g/dL vector-encoded bAT87Q-globin Hemoglobin (g/dL) 6 HbAT87Q 4 HbF HbE 2 HbA 0 0 10 20 30 40 50 60 Months post-transplantation

  10. Partial correction of dyseythropoiesis and red blood cell maturation/lifespan 4,0 35 4,0 3,5 3,5 30 3,0 25 3,0 Blood erythroblasts (x103/µL) Reticulocytes (x105/µL) Red blood cells (x106/µL) 2,5 2,5 20 2,0 15 2,0 1,5 1,5 10 1,0 5 1,0 10 20 30 40 50 60 10 20 30 40 50 60 Months post-transplantation Months post-transplantation

  11. Partial decrease in plasma ferritin levels Phlebotomies 3500 3000 2500 Ferritin (µg/L) 2000 1500 1000 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 Months post-transplantation

  12. Sustained amelioration of the thalassemic phenotype Complete MCH correction (28.4 pg) (Average MCH for βE/β0-Thal patients = 19 pg) Calculated expression βAT87Q > 70% endogenous βA-globin on a per gene basis Calculated increased RBC lifespan > 8.5-fold Decreased circulating erythroblasts > 3-fold (> 9-fold for vector+ cells) Significant decrease (> 2-fold) in plasma ferritin levels Excellent clinical status (full time job)

  13. 100% 80 60 40 20 0 3 5 9 13 16 18 19 20 24 Relative dominance of IS at the HMGA2 locus (dominance relative to other IS, but > 80 % cells remain untransduced) Integration site (IS) analysis by LM-PCR and DNA pyrosequencing (whole nucleated blood cells and purified sub-populations) Low total number of different IS (< 300) In actively transcribed regions, similar to generic HIV vector HMGA2 FBXL11 TBC1D5 PILRB MKLN1 IRAK1 ZZEF1 RFX3 HMGA2 NUP98 ATXN10 EPB41L2 EIF1 PHF16 SAE1 GNA12 POLA2 Months post-transplantation (whole nucleated blood cells) 24 IS both myeloid and lymphoid

  14. Aberrant splicing of the third intron and polyadenylation within the globin lentiviral vector Let-7 miRNAs Globin-LV ATG TAG 51516 51517 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 +++++

  15. Cell type distribution of HMGA2 IS (3 months post-transplant) CFU-GM colonies ~ 7 % positive (~ 67% of vector+) BFU-E colonies ~ 15 % positive (~ 78% of vector+) Lymphocytes (thorough search) ~ undetectable (fresh sorted CD3 / CD19 or lectin expanded) LTC-IC (7 weeks) ~ 6 % positive (~ 50% of vector+)

  16. First evidence in humans for long-term hematopoietic lineage bias (myleoid vs. lymphoid vs. totipotent) Further evidence is accumulating in mice … • By limited dilution transplant analyses from C. Eaves lab (e.g., Cell Stem Cell 2012;10:273-83). • By barcoded transplant analysis from P. Leboulch lab (submitted). • CD86- marks myeloid restriction from W. Kincade lab (Blood 2012;119:4889-97).

  17. 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 No abnormality detected for MPL. JAK2 and TET2 Normal LTC-IC counts No preferred engraftment in NSG mice (coll. C. Eaves) Asymptotic stabilization of the clone relative dominance Natural cases of PNH with HMGA2 dysregulation followed for > 19 years

  18. HMGA2 IS is frequently retrieved by DNA pyrosequencing in vivo after retroviral and lentiviral human CD34+ gene transfer HMGA2 in X-SCID trial (γ-RV vector) • > 15 cluster IS in HMGA2 (aggregates of patients data): • - several in HMGA2 Intron 3 • - several with tendency to increase with time and then stabilize • - 2 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 Wiskot Aldrich trial (γ-RV vector) MGMT glioblastoma trial (γ-RV vector) - Lack of detection in mouse studies - Evidence for initial integration bias ? - Evidence for homeostatic in vivo selection ? - General principle seen with other key genes

  19. The percentage of partially dominant HMGA2 site is decreasing over time in whole nucleated blood cells (qPCR – 5 years post-transplant)…

  20. Integration site (IS) analysis by LM-PCR and DNA pyrosequencing 4 and 5 years post-transplantation 4 years 5 years post-transplantation 100% 90% 80% 70% 60% Insersion sites (% of each) 50% 40% 30% 20% 10% 0% WBC WBC CD45 CD3 CD19 CD15 CD14 Erythro Number unique IS 38 27 20 10 22 19 23 9 SPATS2 POLA2 ZZEF1 Already the most common after 3 years HMGA2 RFX3 PMS2P1

  21. qPCR based kinetics of the four most common IS up to 5 years post-transplantation 0.14 copies 0.12 HMGA2 0.10 RFX3 0.08 FBXL11 Vector copy number 0.06 ZZEF1 0.04 0.02 0.00 0 10 20 30 40 50 60 Months post-transplantation 4 years ZZEF1 = 1/3 HMGA2 5 years ZZEF1 = HMGA2

  22. Second E/0-thalassemia (major) gene therapy patient transplanted on November 24, 2011 • 23 year old woman, βE/β0‑Thal Major • Transfusion dependent since her 2nd month of life. No HLA matched sibling donor • The transplantation was uneventful.Engrafted neutrophils by day 20 and had • delayed platelet reconstitution (no related complications) • The patient has returned to full time work • Peripheral vector copy numbers and therapeutic βAT87Q‑globin protein in reticulocytes • approximately 3-fold greater than in the previous patient at same time-points • Red blood cell transfusions are now being tapered down to stimulate erythropoiesis • and increase βAT87Q‑globin production

  23. Parallel evolution of therapeutic Hb in the second β-thalassemia patient after lentiviral gene therapy PATIENT 1 (specific chain / non-a chain) PATIENT 1 20 1.6 1.4 [g/dL] 15 1.2 [%] b87 b87 1.0 g g bE bE 10 0.8 globins hemoglobins d d 0.6 5 0.4 0.2 0 0.0 g bE 0 2 4 6 8 0 2 4 6 8 Months post transplant Months post transplant PATIENT 2 (specific chain / non-a chain) PATIENT 2 20 1.6 1.4 [g/dL] 15 1.2 [%] b87 b87 1.0 g g bE bE 10 0.8 globins hemoglobins d d 0.6 5 0.4 0.2 0 0.0 g bE 0 2 4 6 8 0 2 4 6 8 Months post transplant Months post transplant

  24. Comparative globinchainanalysis by reverse phase HPLC Patient 1 (transfusion independent since 4.5 years ago) and Patient 2

  25. Integration site (IS) analysis by LM-PCR and DNA pyrosequencing in Patient 2 “MHV” 6 months post-transplantation CD34 WBC CD45 CD3 CD19 CD15 CD14 Erythro 100% 90% 80% 70% 218 unique sites 60% Insertion sites (% of each) 50% 40% 30% 20% 10% 0% VPS16 DKFZP434 C2019 JUP PLEKHA3 TCF12* IMAA GTF2IRD2 SLC19A2 BC031827 DHDDS GUSBL2 VAPA GTF3C2 ZNF318* C12ORF64 CIP29* ARID1A* GUSBL2 LHX3 IL6ST* ATP6V1A SF3A3 DKFZP434 N1217 BST2 NBR1* MAG BTBD11 TXNDC11 ZNF280B DHX8 HORMAD2 CLN3 FBXL11(3) NR_002821 UBA2,SAE2 NHN1 CEP290 VWF SAPS3 KIF1B low NUP88 PTRF FLJ45340 CARM1 TK1* FBXL11(2) RHOA* CRAMP1L TNFSF12 PFAS FCHSD2 RAB14 CUTL1 PB1 TNKS2 NCK1* CEP110* SUV420H1 MIER2 HMGA2* MPRIP SAFB ARIH1 KIAA0827 PLEKHA3 PMS2P1 FBXL11(1) AK090973 B7 C9ORF86 SPATS2 MALAT1 C4ORF45 CHD6,CHD5* C11ORF49 MAP1S VDP MEX3C C19ORF47 SETD2* DKFZP686B0790 C17ORF26 MAP3K3 CSNK1G1 SPOCK3 GATAD2B TAFII105 DNAL1 UNQ747 low ACSL4 EIF4G3 TXNRD3 RPS6KA1* low WDHD1 RAB5C* C6ORF106 RBM5* JAK3* SIRT3 IQGAP1 TBLR1 low low low SPOP NAV3 NIP30 low

  26. Modified vector BB305 (G. Veres & M. Finer – bluebird bio) for continuation French trial and for US trial • Removal insulator and TAT independent • together with novel purification system • Higher GMP production titers and >2-3-fold better human CD34+ transduction efficiency • Greater vector copy number (VCN) per cell • (important for β0-thal) • High βAT87Q‑globin protein expression even in non-thalassemic erythroid cells (e.g., βS/ βS) • Maintained low genotoxic potential in IVIM assays (coll. C. Baum) • Vector substitution in French trial planned for next patients • US trial with new vector filed with RAC (unanimous approval) and with FDA in December VCN ~ 3

  27. Institute of Emerging Diseases and Innovative Therapies – INSERM U. 962 – University Paris 11 Bluebird bio – France and Harvard Medical School, Brigham and Women’s Hospital, Boston, MA Emmanuel Payen Olivier Negre Leila Maouche-Chrétien Alisa Tubsuwan Soumeya Abed Robert Pawliuk Karen Westerman Resy Cavallesco Julian Down Kathy Hehir Philippe Leboulch Hopital Necker (AP-HP), Paris Marina Cavazzana-Calvo (current PI clinical trial) Salima Hacein-Bey Abina Olivier Hermine Felipe Suarez Antoine Ribeil Bluebird bio, Inc, Cambridge, MA and bluebird bio - France Olivier Negre Gabor Veres Maria Denaro Béatrix Gillet-Legrand Yves Beuzard Kathleen Kirby Robert Kutner Dave Davidson Mitchell Finer Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de Paul Eliane Gluckman Françoise Bernaudin Gérard Socié University of Pennsylvania School of Medicine, Philadelphia, PA Frederick Bushman Gary Wang Troy Brady German Cancer Research Center (DKFZ), Heidelberg, Germany Annette Deichmann Manfred Schmidt Kristof Von Kalle Indiana Vector Production Facility, Indianapolis, IN Ken Cornetta Scott Cross Chris Ballas Terry Fox Laboratory, University of British Columbia, Vancouver, Canada Melanie Kardel Alice Cheung Connie Eaves Mahidol University and Ramathibodi Hospital, Bangkok, Thailand Alisa Tubsuwan Suradej HongengSuthat Fucharoen

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