Gene therapy in rare anemias

Gene therapy in rare anemias Jose Carlos Segovia Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division CIEMAT-CIBER / IIS-FJD Madrid - Spain 6 th EUROPEAN SYMPOSIUM ON RARE ANAEMIAS 1 st Dutch-Belgian meeting for patients and health professionals 21 st - 22 nd November 2015 Amsterdam - The Netherlands

Disclosures Company name Research support Employee Consultant Stockholder Speakers bureau Advisory board Other

This talk is applicable for: Thalassemia s Sickle cell disease Membrane disorders (e.g. sferocytosis) Enzym defects (e.g. PKD, G6PD) PNH Other forms of hemolytic disease Definite X X X Probable X X

What is the aim of a Gene Therapy? To introduce in the cell nucleic acids with the correct information that will allow, transiently or permanently, the expression of the protein of interest The wild type version of the defective protein that produces de erythroid disease

How does ex vivo gene therapy work? 1. Cells are extracted from the patient 2. A virus is modified to avoid its reproduction maintaining its ability to enter the cell (vector) Gene 3. The gene of interest (healthy PKLR) is inserted in the vector 6. Modified cells are injected in the patient and start to produce the healthy protein Modified cell 4. Patient cells and modified viruses are put in contact 5. Gene modification (correction) occurs in the cells

Which are candidate diseases for gene therapy? Monogenic diseases: Mutations in one known gene generate the disease Recessive inheritance: Both copies of the gene are mutated Allogeneic bone marrow transplantation cures the disease

Which cells should be modified? Erythrocytes / Red Blood Cells Short half-life: 120 days Need to be replenish Hematopoietic Stem Cell (HSC) - Lives all life long - Generates new HSC - Its job is to produce all blood cells - They are in the Bone Marrow but can be moved to the blood

How does it work? Viral vector carrying the healthy RPK gene Defective erythrocytes Healthy erythrocytes

The gene therapy drug AUTOLOGOUS HEMATOPOIETIC STEM CELLS CARRYING THE CORRECTED GENE Healthy RPK gene How can it be tested?

Mouse model

Mouse model for Pyruvate Kinase Deficiency Erythrocyte parameters Healthy mouse Mouse strain PKD mouse RBC 10,5 x 10 12 /L 6,39 x 10 12 /L HGB 13,8 g/dl 9,7 g/dl HCT 46,2% 38,9% Healthy PKD Min-Oo et al. Nature Genetics (2003) Healthy PKD

The treatment Chemo- and/or Radio-therapy Are they corrected? Autologous transplantation

The results: No anemia 14 Erythrocytes (1x10 12 /L) 12 10 8 6 4 2 0 Mouse: 1 2 3 4 WT B19 B19 B19 Vector: ----- ----- EGFP RPK 30 dpt 60 dpt 90 dpt Meza N et al, Molecular Therapy (2009)

% of Reticulocytes The results: No reticulocytosis 30 25 20 15 10 30 dpt 60 dpt 90 dpt 5 0 1 2 3 4 Mouse: WT B19 B19 B19 Vector: ----- ----- EGFP RPK Meza N et al, Molecular Therapy (2009)

The results: Late progenitors balance correction Progenitores eritroides PROERYTHROBLASTS BASOPHILIC ERYTHROBLASTS POLYCHROMATOPHILIC ERYTHROBLASTS ORTHOCHROMATIC ERYTHROBLASTS RETYCULOCYTES AND ERYTHROCYTES Meza N et al, Molecular Therapy (2009)

Erythropoietin levels estabilization E r ythroid values of control and transplanted RPK deficient mice Animals Hg level (g/dl) Hct MCV (fl) Plasma Fe level ( m g/dl) Plasma erytrthropoietin level (pg/dl) WT 13.6 0.75 47.8 5.06 45 2.0 251.8 62.5 20.8 10.22 B19 8.7 1.06 33.3 2.51 54 0.70 94.8 6.04 163.7 16.45 EGFP 9.1 0.7 32.4 1.89 52.5 1.27 95.3 9.29 144.7 14.48 RPK 12.21 0.51 43.75 2.43 43.42 1.51 220.2 65.7 14.7 14.17 Meza N et al, Molecular Therapy (2009)

The results: No splenomegaly Transplanted with corrected cells Healthy PKD Spleen morphometry Transplanted with corrected cells B19 Healthy Meza N et al, Molecular Therapy (2009) 0 50 100 150 200 250 300 350 Spleen weight (mg)

Reduction of iron deposits Meza N et al, Molecular Therapy (2009)

Has gene therapy been applied in humans?

Gene therapy clinical trials in hematopoiesis Kaufman et al, EMBO Mol Med (2014)

β-thalassaemia ex vivo gene therapy Transfusion-independent 12 months after gene therapy Cavazzana-Calvo et al, Nature (2010)

β-thalassaemia ex vivo gene therapy Cavazzana-Calvo et al, Nature (2010)

β-thalassaemia ex vivo gene therapy Hematopoiesis turned to be oligoclonal Cavazzana-Calvo et al, Nature (2010)

New β-globin gene therapy trials Subject Number Age of consent Characteristics of subjects with β-thalassemia major prbc CD34+VCN in Transfusion Cell Sex Genotype drug Requirement source (ml/kg/year) a substance CD34+ Cell Dose (x10 6 /kg) Follow up 1201 18 Female β 0 /β F 139 Apheresis 1.5 8.9 15M 1202 16 Male β 0 /β F 188 Apheresis 2.1 13.6 15M Not yet 1203 19 Male β 0 /β 0 176 Apheresis 0.8 treated Indication for transplant Age of Consent Characteristics of subject with severe sickle cell disease (1204) prbc CD34+VCN in Transfusion Cell Sex Genotype drug Requirement source substance (ml/kg/year) Multiple VOCs 13 Male β 0 /β 0 179 ACS Silent cerebral infarct X CD34+ Cell Dose (x10 6 /kg) Follow up Bone Marrow 1.2/1.0 5.6 6M a Mean prbc requirement per year, over the past 2 years prior to consent b VCN = number of vector copies per diploid genome c ACS = acute chest syndrome Cavazzana-Calvo et al, EHA Meeting (2015)

Is it safe?

Gene therapy clinical trials in hematopoiesis Target cell/injection Disease Transgene Vector HSC SCID-X1 IL2Rγc Gammaretroviral WAS WASP Gammaretroviral Modified from Kaufman et al, EMBO Mol Med (2014)

What about PKD?

Orphan Drug Designation in August, 2014 EU/3/14/1130 Lentiviral vector carrying the healthy RPK gene Healthy RPK gene 1 Design and development of the THERAPEUTIC VIRUS 4 ORPHAN DRUG designation Registered drug 6 Gene therapy development 2 In vitro validation 3 Preclinical studies in mouse models (EFFICACY and SAFETY) 5 CLINICAL TRIALS

ENERCA The consortium PATIENTS SIDE DRUG SIDE P2: R van Wijk UMCU Utrecht- The Netherlands P4: JL Vives HClinB Barcelona - Spain P6: S Pissard INSERM Paris - France P3: P Bianchi IRCCS Milan - Italy P5: L Ribeiro CHUC Coimbra - Portugal P7: I Badell HPS Barcelona - Spain P1: JC Segovia CIBER Madrid - Spain P8: J Sevilla SERMAS Madrid - Spain P9: GMP viral production facility P11: J Mountford Univ Glasgow Glasgow UK P10: Farzin Farzaneh KCL London UK VIRAL VECTOR PLATFORM EU PKD Patient s groups / Spanish Association for Rare Diseases

Who could be enrolled in the Clinical Trial? French study (preliminary results) No data 12 % (7) Never transfused 30 %(18) Continuous Transfusion 23 %(14) No Surgery 36 % (5) No Surgery 48 %(10) Surgery Spleen surgery 64 % (9) Transfusion ended 35 %(21) Surgery 52 % (11) Courtesy of S. Pissard, Paris France Medium value of post surgery increase in Hb : 2 g/dl

Patient s support www.pyruvatekinasedeficiency.com

Patient s support

Patient s support 832 answers UK; 35 Venezuela; 1 USA; 135 Australia; 44 Belgium; 38 Canada; 27 Cyprus; 1 Denmark; 81 Ecuador; 1 The Netherlands; 93 France; 125 Sweden; 1 Spain; 164 Saudi Arabia; 1 Rumania; 1 Portugal; 4 Germany; 1 Greece; 7 Italy; Mexico; 62 1 Ireland; 2 Norway; 1 Latvia; 1 Pakistan; 1 Poland; 1 New Zealand; 3

Patient s support I'm a patient with PKD 4% (37) Link to PKD Other 23% (195) I'm a family member of someone with PKD 16% (131) I'm a parent of a child/adult with PKD 6% (48) I'm a friend of someone with PKD 51% (421)

Susana Navarro M. García-Gómez Raquel Chinchón Sergio López Differentiation and Cytometry Unit María G. Bravo Israel Orman José Bustos Oscar Quintana Sara Fañanás Rebeca Sanchez Omaira Alberquilla

Acknowledgements Juan A. Bueren Hematopoietic Innovative Therapies King s College London (London) M. Antoniou Agios Pharmaceuticals (Boston) Sh. Jin C. Kung P. Kosinski HSR-TIGET (Milan) E. Montini A. Calabria F. Benedicenti Hannover University A. Schambach H. Clinic (Barcelona) J.L. Vives-Corrons M. Mañu PKreset Ca Granda OMP (Milan) P. Bianchi A. Zanella H.U. Utrecht (Utrecht) R v. Wijk E.J. v. Beers H.I.U. Niño Jesús (Madrid) J. Sevilla H. Sant Pau (Barcelona) I. Badell INSERM (Paris) S. Pissard Hospitals L. Riveiro (Portugal) MedCare CIEMAT www.pyruvatekinasedeficiency.com Laure Gelsin Patients and Healthy donors EpiSevi 2.0

Gene therapy clinical trials in hematopoiesis Target cell/injection Disease Transgene Vector HSC SCID-X1 IL2Rγc Gammaretroviral WAS WASP Gammaretroviral Modified from Kaufman et al, EMBO Mol Med (2014)

Target patients (PKD Natural History Study) 6/75 EHA Learning Center. Grace R. Jun 12, 2015; 100615