Pluripotent stem cell based gene therapy for hematological diseases Stijn Vanhee, Bart Vandekerckhove  Critical Reviews in Oncology / Hematology  Volume 97, Pages 238-246 (January 2016) DOI: 10.1016/j.critrevonc.2015.08.022 Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions

Fig. 1 Hematopoietic stem cell based gene therapies for hematological diseases. (A) Standard gene therapy using retroviral or lentiviral transduction of hematopoietic stem progenitor cells (HSPC). Patient derived HSPC are transduced with an expression vector encoding the functional gene. Upon successful transduction, gene substituted HSPC are generated; (B) gene therapy using tailored nucleases to obtain gene edited HSPC. Critical Reviews in Oncology / Hematology 2016 97, 238-246DOI: (10.1016/j.critrevonc.2015.08.022) Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions

Fig. 2 Different methods for gene editing. Tailored nucleases such as zinc finger nucleases (ZFN), tale like effector nucleases (TALEN), or alternatively the CRISPR/Cas9 system, can be used for genome editing. ZFN consist of generally 4 zinc finger motives, each recognizing 3 bp, linked to a FokI nuclease. TALEN on the other hand consist of TALE proteins, each recognizing a single nucleotide, linked to a FokI nuclease protein. Upon DNA binding of the ZFN or TALEN, the FokI nucleases are in close proximity, dimerize and catalyze a double stranded break (DSB). The Cas9 protein, on the other hand, is targeted towards the specific gene by a single guide RNA (sgRNA) that is complementary to a 20 bp stretch in the genomic DNA. This Cas9 protein induces a DSB with high efficiency. When a repair template is introduced, the DSB can be corrected through homologous recombination (HR), inserting the corrected gene fragment orthotopically. Critical Reviews in Oncology / Hematology 2016 97, 238-246DOI: (10.1016/j.critrevonc.2015.08.022) Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions

Fig. 3 Pluripotent stem cell based gene therapy for hematological diseases. Gene therapy uses tailored nucleases to obtain gene edited hematopoietic cells derived from patient specific pluripotent stem cells. Somatic cells are reprogrammed towards induced pluripotent stem cells (iPSC), in which the affected gene is then corrected. After in vitro hematopoietic differentiation of iPSC, the obtained cells can be transfused back into the patient. Critical Reviews in Oncology / Hematology 2016 97, 238-246DOI: (10.1016/j.critrevonc.2015.08.022) Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions

Fig. 4 schematic overview of hematopoiesis. Comparison of the ontogeny of the hematopoietic system in the mouse with the development of hematopoietic cells from human pluripotent stem cells in vitro. In the murine system, a first wave of primitive hematopoiesis occurs in the yolk sac at embryonic day 7.5 (E7.5). Here primitive erythrocytes (pEry) and primitive megakaryocytes (pMk) are generated. Subsequently a definitive wave of hematopoiesis is initiated between E8.5 and E9.5, during which erythro-myeloid progenitors (EMP) and lympho-myeloid progenitors (LMP) are formed. Transplantable HSPC are generated in the embryo at E10.5 in the aorta-gonado-mesonephros (AGM) region. These cells will then migrate to the fetal liver, where they further develop and expand. In vitro pluripotent stem cell differentiation shows analogy with the in vivo observations in mice. After 5–7 days of differentiation, primitive erythrocytes and megakaryocytes are generated. Later on in culture, between day 10 and 14, erythro-myeloid progenitors (EMP) and lympho-myeloid progenitors (LMP) are generated. Whether these cultures can proceed to the generation of an AGM-like hematopoietic process has not been unequivocally established. Critical Reviews in Oncology / Hematology 2016 97, 238-246DOI: (10.1016/j.critrevonc.2015.08.022) Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions

Fig. 5 Generation of hematopoietic cells from pluripotent stem cells with clinical applicability. Gene corrected induced pluripotent stem cells (iPSC) derived blood cells can be reprogrammed to generate gene corrected induced HSC (iHSC) and transfused into the patient. Alternatively, iPSC can be directly differentiated towards end cells for transplantation: T cells for the treatment of severe combined immune deficiency syndromes, or tissue macrophages for the treatment of Hurler syndrome Critical Reviews in Oncology / Hematology 2016 97, 238-246DOI: (10.1016/j.critrevonc.2015.08.022) Copyright © 2015 Elsevier Ireland Ltd Terms and Conditions