Gene therapy of severe β-thalassemia

Transcription

1 Gene therapy of severe β-thalassemia Yves Beuzard Thalassemia/ENERCA conference, Madrid, November 2010

2 Overview of the Clinical Protocol Autologous transplantation of transduced CD34+ cells Mobilized Blood Cells CD34+ cells Vector + Cytokines Maximize % Transduced HSCs or Testing and Release While Frozen Bone Marrow Harvest Busulfex Bone Marrow Conditioning Maximize Myeloablation IV Infusion Transduced Cells (2x10 8 unsorted BM cells/kg kept for rescue) 2

3 Clinical History of Patient "PL" Male with severe βe/β0-thalassemia. Diagnosis: 1 year old. Fully transfusion dependent since age 3 (225 ml RBCs /kg/year). Spontaneous Hb levels as low as 4.5 g/dl. Major hepato-splenomegaly (splenectomy at age 6) and growth retardation. Failure of Hydroxyurea therapy. Deferoxamine (5 days/week) since age 8. No liver fibrosis. Iron overload by liver MRI (561 µmol/g). No cardiac toxicity. No related, genoidentical HLA-matched donor. Autologous transplantation at age 18, on June 7, 2007 (3.5 years ago)

4 Lentiviral vector design with marked β-globin gene 2 x chs4 Insulator cores 2 x chs4 Insulator cores Ψ + cppt/flap human β-globin gene 266bp 644bp 845bp 1153bp III II I βp HS2 HS3 HS4 ppt RRE 3 enhancer SIN + Insulator (Δ 1 core in 50%) β A-T87Q SIN + Insulator (Δ 1 core in 50%)

5 Conversion to transfusion independence (I) Desferal Exjade Deferiprone Last RBC transfusion 2.5 years ago Phlebotomies (2200 ml) 12 Hemoglobin (g/dl) appendicitis Months post-transplantation RBC transfusions Phlebotomies

6 HPLC analysis of globin chains in whole blood (3 to 33 months post-transplant) Absrobance 220 nm Absrobance 220 nm β A β E β A-T87Q β E β A 3 months α γ G γ A 12 months α γ G γ A Absrobance 220 nm β E βa-t87q β A-T87Q Σ (all β + γ) 33 months α γ G γ A x 100 = % -5.0

7 Conversion to transfusion independence (II) HbA HbA T87Q HbF HbE 10 Hemoglobin (g/dl) HbA HbA T87Q HbF HbE Months post-transplantation

8 Decreased dyserythropoiesis and increased RBC survival Erythroblasts (x10 3 /µl) Decreased circulating erythroblasts Months post-transplantation RBC (x10 6 /µl) Increased RBC lifespan Months post-transplantation Retic (x10 5 /µl) Near normal endogenous level of β A-T87Q -globin expression in circulating RBCs on a per gene basis MCH correction: Patient's MCH months post-transplant = 28.7 pg (within normal range pg) Average MCH for β E /β 0 -Thal patients = 19 pg β A-T87Q -globin β A -globin (normal) = MCH P x Hb A-T87Q (%) x 2 = 0.69 MCH N Gene expression output on a per gene basis = %

9 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 100% IS both myeloid and lymphoid Months post-transplantation (whole nucleated blood cells) HMGA2 HMGA2 FBXL11 TBC1D5 PILRB MKLN1 IRAK1 ZZEF1 RFX3 NUP98 ATXN10 EPB41L2 EIF1 PHF16 SAE1 GNA12 POLA2 Relative dominance of IS at the HMGA2 locus (dominance relative to other IS, but 90% blood cells remain untransduced)

10 Vector integration in the third intron of the HMGA2 gene ATG Globin-LV TAG Let-7 mirnas E1 E2 E3 E4 E5 Intron 3 (~ 113 kb) Deletion of 1 copy insulator core Physiological regulation of HMGA2 expression Expression largely restricted to embryonic and stem cells Normal degradation of RNA by Let-7 mirnas (multiple targets in E5) Decreased expression in HSCs correlated with aging

11 Quantification by qpcr of the number of copies of vector provirus in circulating blood cells (overall and HMGA2 IS) (qpcr for whole nucleated blood cells and purified sub-populations) Whole nucleated blood cells 0.14 Vector copy number Months post-bmt 10-12% 2-4% Total vector copy number (black) and copy number at specific HMGA2 site (blue)

12 Proposed target of Globin LV-HMGA2 IS clone formation HMGA2 IS undetectable in lymphocytes LT-HSC LTC-IC HMGA2 IS 5 % Lymphoid-restricted LT-HSC (?) Myeloid-restricted LT-HSC (?) " α cells" "Stemness" acquisition Common Myeloid Progenitor HMGA2 IS 15 % ΔHMGA2 RNA > 10,000x BFU-E BM Erythroblasts CFU-GM HMGA2 IS 7 % ΔHMGA2 RNA = 0 Erythroblasts Granulo-Mono

13 CONCLUSIONS (I) High expression of therapeutic globin gene % normal endogenous β A -globin on a per gene basis No evidence of secondary repression (PEV) Correction of MCH (19 pg 29 pg/gr) Therapeutic gene contributes # 3.5 g / dl Hb in blood Conversion to transfusion independence for the last 27 months Major increase in survival of corrected RBCs (life span x 8,7) Hb 9-10 g/dl Reduction in reticulocytosis and erythroblastosis Much improved quality of life Excellent tolerance: feeling cured, full time cook. Oral iron chelation + phlebotomies: major decrease in blood ferritin and liver iron levels.

14 CONCLUSIONS (II) HMGA2 Clone in myeloid cells, not present in lymphocytes - Transcriptional activation by LCR, in erythroblasts only. - Failure of single core chs4 insulator in the erythroid precursors, but efficiency in granulocytes/monocytes. - truncation of HMGA2 mrna and loss of Let 7 micro-rna repression Presumptive homeostasis: stable HMG2A2 clone, normal caryocytology, to be monitored for 15 years Hypothesis: Amplification of myeloid-biased LT-HSC with lineage priming Other cases of IS in HMGA2: SCID-X1 (RV), ALD (LV) Benign expression of truncated HMGA2 gene (Lipomas and other benign tumors, PNH) Importance of HSC dose: increased risk vs. increased appearance of polyclonality

15 Institute of Emerging Diseases and Innovative Therapies CEA INSERM U. 962 Universities Paris 7 and 11 and Bluebirdbio - France Emmanuel Payen Olivier Negre Béatrix Gillet-Legrand Floriane Fusil Céline Courne Yves Beuzard Philippe Leboulch Harvard Medical School, Brigham and Women s Hospital, Boston, MA Robert Pawliuk Karen Westerman Resy cavalleso Philippe Leboulch Hopital Necker (AP-HP), Paris Salima Hacein-Bey Abina Laure Cacavelli Marina Cavazzana Calvo (PI) University of Pennsylvania School of Medicine, Philadelphia, PA Gary Wang Troy Brady Frederick Bushman Other University Hospitals of Paris (AP-HP): Saint-Louis, Mondor, CHIC, Tenon, Saint Vincent de Paul Eliane Gluckman (PI) Françoise Bernaudin Gérard Socié Robert Girot Jean Soulier Nabil Kabbara Bluebird Bio, Cambridge, MA Kathy Hehir Maria Denaro Julian Down Indiana Vector Production Facility, Indianapolis, IN Ken Cornetta Scott Cross Chris Ballas

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17 Percentages of vector bearing cells in blood and bone marrow (qpcr 3 years post-transplant) BONE MARROW (36 months) All nucleated cells 21.0 % CD % CD % Erythroblasts (glycoa+ CD71+) 34.6 % BLOOD (35 months) All nucleated cells 8.9 % Granulo-Monocytes (CD15+) 18.6 % B Lymphocytes (CD19+) 9.4 % T Lymphocytes (CD3+) 2.3 % Erythroblasts (CD45- CD71+) 1.1 % 20% Correction dyserythropoiesis Memory T cells not exposed

18 Transduction / transplantation parameters Viral titer cgmp lot = 1.3 x 10 8 TU / ml during CD34 + cell transduction Fresh marrow for transduction and mobilized CD34+ cells for rescue (back up) Pre-transplant transduction efficiency = 0.6 vector copy / CD34 + cell (qpcr) Pre-translant conditioning: BUSULFEX alone (at myeloablative dose) Dose of CD34+ cells injected = 3.9 x 10 6 CD34 + cells / kg

19 Expression of β A-T87Q - globin in whole blood (HPLC analysis) β AT87Q Therapeutic Hb (%) p < r 2 = Hb β AT87Q (g/dl) Months post-transplantation

20 Kinetics of γ-globin expression Ratios blood globins / βe (normalized HPLC Areas) 3,0 2,5 2,0 1,5 1,0 0,5 0, Months post-transplantation βe γa+γg β87

21 Hematopoietic reconstitution with change in blood Granulo-Mono (GM) / Erytho (EB) ratio Platelets / µl EB PLT Neutro GM Months post-transplantation Neutro, GM, EB / µl Kinetics of blood vector-bearing cells Vector copy number Reconstitution Change in GM/ EB ratio Months post-transplantation

22 Integration site (IS) analysis by DNA pyrosequencing (Blood cell subsets) T lymphocytes (CD3+) B lymphocytes (CD19+) Granulocytes Monocytes (CD15+) Erythroblasts (CD45-/CD71+) Dominance relative of the HMGA2 IS in both granulocytes-monocytes and erythroblasts, but not lymphocytes (negative qpcr after lymphocyte expansion)

23 Blood CD15+ cells (GM) % 8-9% Months post-bmt Blood CD45-/CD71+ cells (E) % 1-2% Months post-bmt

24 Is partial clonal dominance linked to HMGA2 activation? Evidence of truncation of the main HMGA2 transcript (E1 E3) by staggered RT-qPCR Sequencing of the main HMGA2 transcript: Aberrant splicing within the vector insulator + polyadenylation

25 LCR-mediated effect vs. loss of Let-7 mirna targets? RT-qPCR "run-on" Only expressed in erythroid cells (not granulo-mono) in spite of similar percentage vector at HMGA2 IS (qpcr HMGA2/LV junction) Increased transcription = 371 fold (likely LCR-driven with insulator failure) Increased RNA stability = 36 fold (likely loss of Let-7 mirna targets)

26 Detection of the HMGA2-derived protein Western blot β-thal individual PLB β-thal individual PLB Rec HMGA2 Truncated Full-length WB: anti-hmga2 20 Detection of the protein in erythroid cells derived from BFUes in vitro Ponceau staining

27 Stabilization of HMGA2 mrna expression in whole blood (erythroblasts) RT-qPCR analysis Red dots: RNA was extracted from whole blood before (0) and after transplantation.

28 Absence of major position effect variegation (PEV) regardless of integration site (IS) dominance Hb A-T87Q (%) Globin HPLC analysis of individual vector-bearing (PCR+) BFU-Es Homogenous -globin expression γ in single BFU-E colonies regardless of vector integration sites (HMGA2 or not) months post-transplantation (during HMGA2 IS dominance) 0 6 months post-transplantation (before HMGA2 IS dominance)

29 Proposed target of Globin LV-HMGA2 IS clone formation HMGA2 IS undetectable in lymphocytes LT-HSC LTC-IC HMGA2 IS 5 % Lymphoid-restricted LT-HSC (?) Myeloid-restricted LT-HSC (?) " α cells" "Stemness" acquisition Common Myeloid Progenitor HMGA2 IS 15 % ΔHMGA2 RNA > 10,000x BFU-E BM Erythroblasts CFU-GM HMGA2 IS 7 % ΔHMGA2 RNA = 0 Erythroblasts Granulo-Mono

30 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 Asymptotic stabilization of the clone relative dominance (in agreement with mathematical modeling and the natural cases of PNH with HMGA2 dysregulation followed for > 17 years) Importance of the HSC dose: - In deterministic mathematical models, increasing transduced HSC dose results in increased risk with paradoxical increase in appearance of polyclonality - In certain stochastic mathematical models, increasing transduced HSC dose results in balanced co-suppression

31 Recent evidence of HMGA2 IS in other gene therapy trials ("hotspot" or evidence of homeostatic in vivo advantage?) 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) Interpretation of polyclonal patterns of IS (ALD, ADA trials) True lack of genetic dysregulation resulting in growth advantage "Peaceful co-existence" of cells with minor genetic dysregulation