Cell therapies to treat haematopoietic disorders

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2 Cell therapies to treat haematopoietic disorders Haematopoietic Stem Cells (CD34) RBCs Platelets Limitations A donor required for each treatment Transfusion-transmitted infection Immunological (HLA/AB0) matching Cancer/Leukaemia Immunological disorders Anaemia Thalasaemias Trauma Surgery Clotting disorders Following BMT Chemotherapy

3 What are the alternatives? Differentiation from Pluripotent Stem Cells ex vivo expansion? in vitro differentiation of CD34

4 not dependent on donors could be free of infection could be immunologically compatible (Oct4/Sox2/Klf4/Myc) Somatic cells Embryonic Stem Cells Induced Pluripotent Stem Cells SELF-RENEW DIFFERENTIATE

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6 to better understand the biology of RBC production to scale-up a manufacturing process using bioreactors to translate into a first-in-man clinical trial (safety assessment) to develop a business plan Funded by:

7 Our role in the project Generate lines from specific blood groups Produce reporter cell lines to track blood cell production and find better culture conditions. Improve the production of blood cells from s by genetic programming using transcription factors.

8 Generation of 0RhD- s Recruit 5 0RhD - volunteers skin biopsy Episomal transfection of OCT4,SOX2 KLF4,MYC Established > 20 lines from 2 individuals Culture dermal fibroblasts (frozen stocks of 4 lines established) Pluripotency Karyotype and SNP analyses Haematopoietic differentiation

9 Characterisation of lines Pluripotency markers Tra-1-60 Oct 3/4 SSEA-4 Differentiation SSEA-1 Sample ID Tra-1-60 (%) Oct3/4 (%) SSEA4 (%) SFCi55 unstained SFCi55 stained Sample ID SSEA1 (%) SFCi55 unstained 0.01 SFCi55 stained 4.01 Genomic analysis Karyotype: 46 XX SNP: no abnormalities detected

10 Differentiation of s into haematopoietic progenitor cells (HPCs) BFU-E CFU-Mix CFU-GM d6 SFCi51 SFCi50 SFCi53 SFCi55 SFCi56 SFCi57 SFCi96 SFCi97

11 A clinical grade protocol of make RED BLOOD CELLS from PSCs Research grade Jo Mountford (University of Glasgow) Clinical grade

12 Challenges in PSC-derived RBCs Low efficiency of enucleation Nucleated RBCs Primitive cells - mainly express embryonic (ζ / ε) and fetal (γ) globins

13 Why don t PSC-derived RBC mature and enucleate? Adult CD34 HPC EP RBC Enucleated red cells What are the differences? PSC nucleated red cells Factors that are missing Stage Adult blood PSC-blood PSC- blood Jo Mountford Jon Manning

14 Forward programming with Transcription Factors. Transcription factors - master regulators of cell fate KLF1?

15 Krüppel-like factor 1 (KLF1) erythroid lineage commitment terminal maturation of red blood cells Globin switching? involved in enucleation c-myb WT KLF1 -/- GATA1 SOX6 BCL11A KLF1 β-globin locus ε Gγ Aγ δ β (Chr11) Embryonic Fetal Adult

16 Inducible Expression Strategy CAG KLF1 ER T2 KLF1 DAPI - tamoxifen - tamoxifen KLF1 DAPI Hemin Tamoxifen K562 KLF1-ERT 2 R328L-ERT 2 tamoxifen

17 Target KLF1-ER T2 into a safe harbour in the genome pzdonor-aavs1 Puromycin- HA-KLF1-ER T2 AAVS1-LA SA 2A Puro Poly A Poly A ER T2 KLF1 HA CAG promoter AAVS1-RA Co-transfect Zinc Finger Nuclease (ZFN) into human s AAVS1-LA ZFN AAVS1-RA Chromosome in human s

18 Physiological levels of expression achieved Adult CD34 - d0 d10 d0 d10 d tamoxifen KLF1-ER T2 endogenous KLF1 LaminB1 Crude nuclear preps

19 Activation of KLF1 enhances erythroid differentiation Tam Tamoxifen 31 ± 12% 32 ± 12% CD71 Tam 27 ± 9% 68 ± 10% Tamoxifen Tamoxifen CD235a

20 .but at the expense of proliferation

21 Activation of KLF1 enhances the detection of enucleated cells CD235a cells (day 31) Tam Tam CD71 Peripheral blood % Enucleation Hoechst Tamoxifen Tam Tam

22 Activation of KLF1 leads to more robust cells Tamoxifen Mechanism of action?

23 Enhanced the expression of some known KLF1 target genes associated with erythropoiesis Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen Tamoxifen

24 HPLC analyses BUT KLF1-activates the expression of embryonic globins. β Gγ α Aγ ε ζ Tamoxifen

25 Conclusions/next steps Universal donor lines generated Established a strategy for inducible genetic programming Not dependent on random integrations so a more applicable strategy for clinical translation Activation of KLF1 enhances erythroid differentiation of s Appears to enhance the primitive erythroid lineage Produces more robust cells an intrinsic or extrinsic effect? Use this approach with other transcription factors that are expressed at low levels in derived blood cells. Identify those that are able to switch on the definitive haematopoietic programme?

26 Cheng-Tao Yang Melany Jackson Richard Axton Rui Ma Jennifer Easterbrook Helen Taylor Sharmin Hadiera Marths Lopez Yrigoyen Antonella Fidanza Brigid Orr University of Glasgow Joanne Mountford Emmanuel Olivier Lamin Marenah SNBTS Marc Turner Kay Samuel NHS-BT Jan Frayne Dave Anstee/Belinda Singleton