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Engineered Stem Cells in Hematologic Diseases

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Engineered Stem Cells in Hematologic Diseases Next Generation Transplantation: Engineered Stem Cells in Hematologic Diseases George Q. Daley Boston Children’s Hospital/ Harvard Medical School/ HHMI Tandem Meetings 2016 Disclosures: Scientific Advisory Boards (consulting fees and equity) MPM Capital, Solasia, Epizyme, Ocata, Raze, True North, Verastem, AthenaHealth Research Funding: Megakaryon Boston Children’s/ Dana Farber Pediatric Stem Cell Transplant 2015: 114 Transplants Leslie Lehmann Christy Duncan Nick Haining Clinical Director Assoc. Clinical Dir. Assoc. Scientific Dir. Alessandra Biffi Director Gene Therapy Steve Margossian Sung-Yun Pai Suneet Agarwal Michelle Lee Christy Ullirch Jen Whangbo Susanne Baumeister Esther Obang Leo Wang Birgit Knoechel Inga Hoffman Autologous Cell Therapy from iPS Cells NSG Transplant Skin or Blood Cell Primary Immune Deficiency Hematopoietic Bone Marrow Failure stem cells Hemoglobinopathy Reprogramming (Oct4/Sox2/Klf4/Myc) ? Gene Repair Diseased iPS cells Healthy iPS cells ES/ iPS Mammalian Embryoidblood development:Bodies Primitive and Definitive= Yolk Sac waves Morphogens? Drugs? Transcription Factors? Strategies for deriving HSCs from HUMAN pluripotent stem cells (ESC/iPSCs) HSC-specific TFs ?? Respecification CD34+43- ESC/iPSC Hemogenic HSC? CD34+ Endothelium CD45+ TFs Myeloid CFUs Specification Morphogens Respecifying blood progenitors from iPSCs hESC/iPSC EB differentiation - BMP4/cytokines CD34+45+ HPCs D0 D4-7 D11+ D14 HSC TFs 7+ days 9 HSC TFs: Transplant HLF ERG Control MYB SOX4 CD34+45+ KLF4 HPCs RORA Microarray HOXA5 HOXA9 HOXA10 6 6 Respecified progenitors mediate short-term engraftment in NSG mice 5 Dox-inducible factors: ERG, HOXA9, RORA, SOX4, MYB (CD34-5F) Cord blood Erythro-myeloid Erythroid only GlyA CD45 CD45++ gate CD45++ gate GLYA+ gate CD19 CD71 CD33 mTer119 7 7 Engrafted erythroid cells undergo globin switching Erythroid engraftment in vivo 125 CD71 t e p i r 100 c g s n a r 75 b t n i b 50 o l G 25 % 0 B F B 1 2 3 4 5 1 2 3 5 E C m m m m m m m m F F F F F B B B 5 5 5 5 5 C C C GlyA SYTO60 BAND3 8 8 RED BLOOD CELL DIFFERENTIATION FROM iPS PROGENITORS IN VITRO ERG, HOXA9, RORA, SOX4, MYB Respecification Stage I Stage II Stage III 14d -DOX CD34+CD45+ DBA 21 days Stage I Stage II Stage IIIa Stage IIIb D9 D14 D18 D18 E M Lee et al. Nature 2015 9 9 Human IPS RBCs circulate after transfusion Bone Marrow BM + Clodronate Peripheral blood 24h (Enucleation) SYTO60 GLA Human RBCs Colony screen of an expanded set of 28 HSC transcription factors GEMM/BFU-E CFU-GM - HSC- specific + HOXA9 100 TFs ERG 95 RORA 68 68 47 47 EB 34+ EAR 13F 42 37 32 26 ERG TCF7L2 16 HOXA9 ETS1 16 RORA SSBP2 SOX4 PRDM16 11 MYB DACH1 5 HOXA5 PBX1 5 KLF5 HIF3A 5 CIITA HDAC9 5 GATA3 NFIA MEIS1 NRIP1 5 THRB EVI1 5 SKAP1 SMARCA1 0 FOXO1 WWTR1 0 NKX2-3 HOXB4 0 11 11 A subset of TFs is sufficient to induce T- and B-lymphoid potential 28/13F EB CD34+ CD34+CD38- Stromal culture +DOX -DOX OP9-DL1: CD4+CD8+ T cells MS-5: CD19+CD20+ B cells 2wks 5wks 28F 13F 13F CD8 T cells CD3 CD4 TCRab TCRgd 28F 13F 13F B cells CD19 CD19 CD11b CD20 CD10 13F-1 further reduction to 7F A screen for epigenetic modifiers that restrict definitive potential Onder et al. Nature (2012). ERG HOXA9 shRNA RORA SOX4 Measure MYB %CD4+CD8+ T cells CD34+ CD45+ OP9-DL1 shRNA Screen identifies H3K9 HMTs as repressors of T cell potential 5F + shLUC 5F+sh1 5F+sh2 5F+sh3 CD8 5F+sh4 5F+sh5 5F+sh6 5F+sh7 CD4 Repression of top candidate enhances B/T potential of respecified iPSC-derived blood lineages 5F + shLUC 5F + sh1-1 5F + sh1-2 CD8 CELLS T CD4 5F + shLUC 5F + sh1-1 5F + sh1-2 CD19 B CELLS CD56 Generating multipotential progenitors from iPSCs CD34+CD45+ IPSCs GMP-like Patient HOXA9/5 “Core” transcription ERG factors RORA SOX4 NFIA and epigenetic MYB DACH1 regulators CMP-like CLP-like Congenital anemias Immunodeficiencies eg SCA, DBA eg RD, SCID Engineered T cells 16 16 Definitive HSCs from Hemogenic Endothelium Yokomizo T & Dzierzak E, Development, 2010; Wang LD & Wagers AJ, Nature Rev Mol Cell Biol, 2011 Bertrand et al., Nature, 2010 Hematopoietic differentiation thru hemogenic endothelium EB differentiation Endothelial-hematopoietic transition (EHT) 0 1 2 3 4 5 6 7 8 +1 2 3 4 5 6 7 bFGF MACS (CD34+) BMP4 EPO VEGF BMP4 BMP4 BMP4 bFGF TPO IL-6 bFGF bFGF bFGF VEGF FLT-3L IL-11 SB VEGF IGF-1 IL-6 IL-3 CHIR SCF IL-11 SHH EPO IGF-1 Ang II SCF Losartan plate on Matrigel human iPS Protocol adapted from: Ditadi et al., Nature Cell Biol, 2015 Missing signals to confer HSC fate on ES/iPS-HE? HE cells Human Embryo Intrafemoral injection of HEHE can engraft sub-lethally irradiated NSG No engraftment… Selecting Transcription Factors for Screening 26 HSC-TFs (including Rafii, Rossi and Daley) SSBP2, ZKSCAN1, DACH1, TGIF2, GATA2, HLF, BCL11A, NRIP1, IRF2, HOXA5, HOXA9, HOXA10, SIX5, EVI1, MAFF, KLF4, LCOR, RUNX1, GFI1, SPI1, FOSB, MEISS1, ERG, RORA, SOX4, MYB HE dHE FL-HSC Gateway Destination 5’LTR 3’LTR PsiPsi RRE TRE2 Cm ccdB HA hEF1α rtTA3 IRES eGFP GFP+ GFP+ Clone factors into Dox-inducible lenti- vector w/o infection Infected HE cells Screening for factors that convert HE to HSCs Library of 26 TFs Engraftment (peripheral blood) 4w 6w 8w 10w 14w At 10 weeks sort out human- derived BM cells iPS-Hemogenic NSG mice Endothelium Weeks HSC, T, B, Myeloid, Erythroid Flow on engrafted cells Multi-lineage marrow engraftment • Transgene detection in engrafted cells hiPSCs hESCs 7 Defined TFs: HE HSC hiPSC (vivo) SPI1, ERG, hESC (vivo) RUNX1, LCOR, ERG, SPI1 HOX SSBP2, Multiple Hox genes ZKSCAN1 MAFF RUNX1, LCOR MYB, RORA, BCL11A, GFI1 CFU Multilineage engraftment in primary recipients Multilineage engraftment in secondary recipients Globin switching in engrafted red blood cells HBG HBB HBE Engraftment with T and B cells DP T cells in Thymus PMA IFNg Serum ELISA detects Human IgM and IgG Deriving HSCs from human PSCs: a combinatorial approach Developmental Biology: Synthetic Biology: mimicking normal ectopic expression of embryonic development transcription factors (TFs) Embryoid body TFs Cytokines HSC- like cells Evo-devo Synbio HE cells In vivo specification Next Generation Transplantation… Transplant Skin or RBCs Blood Cell PLTs Hematopoietic Combined Gene and Cell Therapy stem cells Transfusable blood products http://www.sciencephoto.com/ …clinical trials in 2017 Koji Eto, Harvey Lodish Reprogramming (Oct4/Sox2/Klf4/Myc) Gene Repair Diseased iPS cells Healthy iPS cells Acknowledgements Sergei Doulatov Linda Thuy Vo Rio Sugimura Thorsten Schlaeger Patricia Sousa Len Zon Marcella Cesana Michael YiFen Lu Hu Li, Jim Collins Chen Vanessa Lundin Melissa Kinney Funding: NHLBI UO1 PCBC, NIDDK R24, DORIS DUKE, HHMI .
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