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 Transplant 2015: 114 Transplants

Leslie Lehmann Christy Duncan Nick Haining Clinical Director Assoc. Clinical Dir. Assoc. Scientific Dir. Alessandra Biffi Director 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 Marrow Failure stem cells Hemoglobinopathy

Reprogramming (Oct4///) ?

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 pluripotent stem cells (ESC/iPSCs)

HSC-specific TFs ?? Respecification

CD34+43- ESC/iPSC Hemogenic HSC? CD34+ 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

CD71 SYTO60

BAND3 GlyA

125

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

8 8 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 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 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 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 (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