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Project Structure

The overall work program of the iMACnet project comprises five experimental subprojects, three of which are executed at the MHH. iMACnet also consists of a central coordination (Z) project that is headed by Prof. Thomas Moritz of the Institute of Experimental Hematology at the MHH.

 

 

Overview of Subprojects                                             Overview of Workprogram

 

 

                       

           

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  • Subproject 1: GMP-compatible generation of gene edited patient-specific iPSCs for the production of autologous i-MAC

     

    Subproject 1 aims to establish the GMP-compatible generation of iPSC lines derived from patients with Pulmonary Alveolar Proteinosis (PAP) and Chronic Granulomatous Disease (CGD), followed by gene editing to correct the underlying genetic defect. Subsequently, the corrected iPSC lines and derived progeny cells will undergo extensive analysis to assure genetic integrity and the absence of designer nuclease-mediated off-target mutagenesis. The following objectives are defined for subproject 1:

     

    i) Generation and characterization of PAP and CGD patient iPSC lines

     

    ii) Seamless CRISPR-Cas9 mediated gene editing to genetically correct underlying defect in patient iPSC lines.

     

    iii) Validation of genetic integrity of gene edited iPSC lines and their derived progeny

     

     

    Relevant Publications

     

    1. Kuhn A, Ackermann M, Mussolino C, Cathomen T, Lachmann N, Moritz T (2017).  TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis. Sci. Rep. 7(1):

    2. Dreyer AK, Hoffmann D, Lachmann N, Ackermann M, Steinemann D, Timm B, Siler U, Reichenbach J, Grez M, Moritz T, Schambach A, Cathomen T (2015). TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells. Biomaterials 69:191-200

    3. Rahman SH, Kuehle J, Reimann C, Mlambo T, Alzubi J, Maeder ML, Riedel H, Fisch P, Cantz T, Rudolph C, Mussolino C, Joung JK, Schambach A, Cathomen T (2015). Rescue of DNA-PK signaling and T-cell differentiation by targeted genome editing in a prkdc deficient iPSC disease model. PLoS Genet 11:1-21

    4. Mussolino C, Alzubi J, Fine EJ, Morbitzer R, Cradick TJ, Lahaye T, Bao G and Cathomen T (2014). TALENs facilitate targeted genome editing in human cells with high specificity and low cytotoxicity. Nucleic Acids Res  42:6762-73

  • Principal Investigator

     

     

     

     

    Prof. Dr. phil. Toni Cathomen

    Institute for Transfusion Medicine and Gene Therapy

    Medical Center-University of Freiburg

    Hugstetter Str. 55

    79106 Freiburg

    Email: toni.cathomenuniklinik-freiburg.de

     

     

     

     

  • Subproject 2: Development of genetic safety switches and their validation

     

    Subproject 2 is focused on addressing the safety issues associated with iPSC-based therapeutic approaches. Like advanced therapy medicinal products (ATMPs), iPSC-derived therapies will have to undergo a risk-benefit assessment and strict safety testing before approval of a clinical application as required by regulatory authorities (e.g. PEI, EMA, FDA). Currently, there are very few clinical studies with iPSC, and potential side effects of iPSC-derived therapies in humans have not been carefully analyzed. As a quality criterion for pluripotency, human iPSC are routinely tested in teratoma formation assays, underlining their capability to differentiate into cells of all three germ layers. It is, therefore, conceivable that these teratoma initiating cells (TIC) – also as a contamination of iPSC in a differentiated cell product – may represent a major safety concern for the application of iPSC-derived cellular therapies. Consequently, subproject 2 hypothesizes that a carefully developed genetic switch will entirely avoid teratoma formation and /or severe adverse events associated with the iPSC-derived cell therapy product. The main objectives of subproject 2 are as follows:

     

    i) To establish and validate a genetic suicide safety switch to remove iPSC.

     

    ii) To establish and validate a genetic suicide safety switch for the induced removal of iPSC and iPSC-derived differentiated progeny.

     

    Relevant Publications

     

    1. Hoffmann, D, Göhring, G, Heuser, M, Ganser, A, Schambach, A*, and Morgan MA* (2015). Production of Mature Healthy Hematopoietic Cells from Induced Pluripotent Stem Cells Derived from an AML Diagnostic Sample Containing the t(8;21) Translocation. Stem Cells [Epub ahead of print] * co-corresponding authors

    2. Hacein-Bey-Abina, S, Pai, SY, Gaspar, HB, Armant, M, Berry, C C, Blanche, S, Bleesing, J, Blondeau,, de Boer, H, Buckland, K F, Caccavelli, L, Cros, G, De Oliveira, S, Fernández, K. S, Guo, D, Harris, C. E, Hopkins, G, Lehmann, L. E, Lim, A, London, W. B, van der Loo, J. C, Malani, N, Male, F, Malik, P, Marinovic, M A, McNicol, AM, Moshous, D, Neven, B, Oleastro, M,Picard, C, Ritz, J, Rivat, C, Schambach, A, Shaw, KL,Sherman, EA, Siberstein, LE, Six, E, Touzot, F, Tsytsykova, A, Xu-Bayford, J, Baum, C, Bushman, FD, Fischer, A, Kohn, DB, Filipovich, AH, Notarangelo, LD, Cavazzana, M, Williams, DA and Thrasher, AJ (2015). A modified g-retrovirus vector for X-linked sever combined immunodeficiency. New England Journal of Medicine 9, 1407-17 

    3. Dreyer AK, Hoffmann D, Lachmann N, Ackermann M, Steinemann D, Timm B, Siler U, Reichenbach J, Grez M, Moritz T, Schambach A, Cathomen T (2015). TALEN-mediated functional correction of X-linked chronic granulomatous disease in patient-derived induced pluripotent stem cells. Biomaterials 69:191-200

    4. Rahman SH, Kuehle J, Reimann C, Mlambo T, Alzubi J, Maeder ML, Riedel H, Fisch P, Cantz T, Rudolph C, Mussolino C, Joung JK, Schambach A, Cathomen T (2015). Rescue of DNA-PK signaling and T-cell differentiation by targeted genome editing in a prkdc deficient iPSC disease model. PLoS Genet 11:1-21

  • Principal Investigator

     

     

     

    Prof. Dr. med. Axel Schambach, Ph.D

    Institute of Experimental Hematology

    Hanover Medical School

    Carl-Neuberg-Straße 1

    30625 Hannover

    Email: schambach.axelmh-hannover.de

  • SubprojectSubproject 3: Scalable generation of pluripotent stem cell derived i-MAC using bioreactor technology under GMP-compliant conditions

     

    Although the generation of human iPSC (hiPSC) derived macrophages has been demonstrated to be feasible, substantial limitations with respect to sufficient cell numbers, quality and/or functionality have been reported. Moreover, the overall generation of i-MAC under GMP-compliant conditions remains elusive. Subproject 3 is aimed at the development of a novel process for the efficient production of i-MAC via continuous suspension culture in stirred, fully instrumented tank bioreactors. The main objectives of subproject 3 are as follows:

     

    i) Establishment and optimization of GMP-compliant expansion of hiPSC as cell aggregates in suspension culture suitable for hematopoietic differentiation

     

    ii) Establishment of continuous hematopoietic differentiation on small-scale suspension culture and stepwise transfer into bioreactors under GMP-compliant conditions

     

    Relevant Publications

     

    1. Zweigerdt, R, Olmer, R, Singh, H, Haverich, A, and Martin, U (2011). Scalable expansion of human pluripotent stem cells in suspension culture. Nature protocols 6, 689-700

    2. Kempf, H, Kropp, C, Olmer, R, Martin, U, and Zweigerdt, R. (2015) Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nature protocols 10, 1345-1361

    3. Lachmann, N., Ackermann, M., Frenzel, E., Liebhaber, S., Brennig, S., Happle, C., Hoffmann, D., Klimenkova, O, Luttge, D, Buchegger, T, Kuhnel, M P, Schambach, A, Janciauskiene, S, Figueiredo, C., Hansen, G, Skokowa, J, and Moritz, T (2015). Large-scale hematopoietic differentiation of human induced pluripotent stem cells provides granulocytes or macrophages for cell replacement therapies. Stem cell reports 4, 282-296

    4. Lachmann, N, Happle, C, Ackermann, M, Luttge, D, Wetzke, M, Merkert, S, Hetzel, M, Kensah, G., Jara-Avaca, M, Mucci, A, Skuljec, J, Dittrich, A M, Pfaff, N, Brennig, S, Schambach, A, Steinemann, D, Gohring, G, Cantz, T, Martin, U, Schwerk, N, Hansen, G, and Moritz, T (2014). Gene correction of human induced pluripotent stem cells repairs the cellular phenotype in pulmonary alveolar proteinosis. American journal of respiratory and critical care medicine 189, 167-182

     

  • Principal Investigators

      

    Dr. rer.nat. Robert Zweigerdt

    Department of Cardiac, Thoracic, Transplantation and Vascular Surgery,

    Hanover Medical School

    Carl-Neuberg-Straße 1

    30625 Hanover

    Email: Zweigerdt.Robert@mh-hannover.de

     

    PD Dr. Nico Lachmann

    Institute of Experimental Hematology,

    Hanover Medical School

    Carl-Neuberg-Straße 1

    30625 Hanover

    Email: lachmann.nicomh-hannover.de

     

    Prof. Dr. phil. nat. Dr. med. Ulrike Köhl

    Institute of cellular Therapeutics and GMP Core Facility IFB-Tx,

    Hanover Medical School

    Carl-Neuberg-Straße 1

    30625 Hanover

    Email: koehl.ulrikemh-hannover.de

  • Subproject 4: Feasibility, safety, and therapeutic efficacy of i-MAC-based pulmonary macrophage transplantation (PMT) in an in vivo model of hereditary Pulmonary Alveolar Proteinosis

     

    Hereditary Pulmonary Alveolar Proteinosis (herPAP) is a rare congenital disease that arises due to mutations in the GM-CSF receptor genes (CSF2RA/B) that lead to a block of alveolar macrophage (AM) differentiation and an inability of AMs to clear alveolar spaces from proteins and phospholipids. Affected children develop massive alveolar protein accumulation, life-threatening respiratory insufficiency and recurrent infections. The goal of subproject 4 is to translate the innovative Pulmonary Macrophage Transplantation (PMT) (1,2) based concept into an effective i-MAC-based treatment approach for herPAP. This work intends to prove the feasibility, safety, and therapeutic efficacy of i-MAC-based PMT in an already established humanized in vivo disease model (huPAP mice). Subproject 4 is based on the hypotheses that (i) i-MAC represent a cell source with considerable plasticity which, upon PMT, will develop into functional alveolar macrophages capable of clearing surfactant factor and proteins from the alveolar space, and (ii) the huPAP model adequately reflects the herPAP disease phenotype as well as the therapeutic effects of the i-MAC-based therapeutic product. The following objectives will be pursued:

     

    i) Standardization of the huPAP-model as a reliable read-out system to evaluate feasibility, safety and efficacy of i-MAC-based PMT

     

    ii) Establishment of the feasibility and safety of i-MAC-based PMT

     

    iii) Establishment of the therapeutic efficacy of i-MAC-based PMT

     

    Relevant Publications

     

    1. Kleff V, Sorg UR, Bury C, Suzuki T, Rattmann I, Jerabek-Willemsen M, Poremba C, Flasshove M, Opalka B, Trapnell B, Dirksen U, Moritz T (2008). Gene therapy of ßc-deficient pulmonary alveolar proteinosis (bc-PAP): Studies in a murine in vivo model. Mol Ther 16: 757-64

    2. Happle C*, Lachmann N*, Skuljec J, Wetzke M, Ackermann M, Brennig S, Mucci A, Jirmo AC, Groos S, Mirenska A, Hennig C, Rodt T, Bankstahl JP, Schwerk N, Moritz T*, Hansen G* (2014). Pulmonary transplantation of macrophage progenitors as effective and long-lasting therapy for hereditary pulmonary alveolar proteinosis. Sci Transl Med, 6:250ra113 *contributed equally

    3. Suzuki T, Arumugam P, Sakagami T, Lachmann N, Chalk C, Sallese A, Abe S, Trapnell C, Carey B, Moritz T, Malik P, Lutzko C, Wood RE, Trapnell BC (2014). Pulmonary macrophage transplantation therapy. Nature, 514:450-4

    4. Lachmann N*, Happle C*, Ackermann M, Lüttge D, Wetzke M, Merkert S, Hetzel M, Kensah G, Jara-Avaca M, Mucci A, Skuljec J, Dittrich AM, Pfaff N, Brennig S, Schambach A, Steinemann D, Göhring G, Cantz T, Martin U, Schwerk N, Hansen G*, Moritz T* (2014). Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis. Am J Respir Crit Care Med, 189:167-82 * contributed equally 

     

     

  • Principal Investigators

     

     

      

     

    Prof. Dr. med. Thomas Moritz

    RG Reprogramming and Gene Therapy

    Institute of Experimental Hematology

    Hannover Medical School

    Carl Neuberg-Straße-1

    30625, Hannover

    Email: moritz.thomasmh-hannover.de

     

     

    Prof. Dr. med. Gesine Hansen

    Clinic for Pediatric Pulmonology, Allergology and Neonatology

    Hannover Medical School

    Carl-Neuberg-Straße 1

    30625 Hannover

    Email: hansen.gesinemh-hannover.de

  • Subproject 5: Curing colitis in chronic granulomatous disease with iPSC-derived corrected macrophages

     

    Chronic granulomatous disease (CGD) is a monogenetic immunodeficiency disorder characterized by defects in gp91phox, p47phox, p67phox or p40phox. These proteins form the subunits of the NADPH oxidase complex, which mediates the formation of the reactive oxygen species (ROS), and thus, antimicrobial activity in myeloid cells. Accordingly, CDG patients are susceptible to bacterial and fungal infections. Next to this, a third of CGD patients develop colitis that, on the histological level, is similar to Crohn´s disease or ulcerative colitis. Based on previous studies, aberrant regulation in intestinal macrophages is thought to underlie immunodysregulation in CGD. Subproject 5, therefore, hypothesizes that (a) severe colitis in CGD is caused by aberrant signaling in lamina propria macrophages which are polarized towards an inflammatory M1 phenotype and (b) iPSC-derived, functionally corrected macrophages (i-MAC) delivered i.v. settle in the lamina propria and down-modulate inflammation. The specific objectives of subproject 5 are:

     

    i) To improve colitis in CGD mice with NADPH oxidase-proficient mouse i-MAC

     

    ii) To treat colitis in NOD/SCID mice with human i-MAC

     

    Relevant Publications

     

    1.Pannick, U, Baumann B, Fuchs S, Henneke P, Rensing-Ehl A, Rizzi M, Janda A, Hese K, Schlesier M, Holzmann K, Borte S, Laux C, Rump EM, Rosenberg A, Zelinski T, Schrezenmeier H, Wirth T, Ehl S, Schroeder ML, and Schwarz K (2013). Deficiency of innate and acquired immunity caused by an IKBKB mutation. The New England journal of medicine 369, 2504-2514

    2. Feuerstein R, Seidl M, Prinz M, and Henneke P (2015). MyD88 in macrophages is critical for abscess resolution in staphylococcal skin infection. Journal of immunology 194, 2735-2745

    3. Klose CS, Blatz K, d'Hargues Y, Hernandez PP, Kofoed-Nielsen M, Ripka JF, Ebert K, Arnold SJ, Diefenbach A, Palmer E, and Tanriver Y (2014). The transcription factor T-bet is induced by IL-5 and thymic agonist selection and controls CD8alphaalpha(+) intraepithelial lymphocyte development. Immunity 41, 230-443 

    4. Klose CS, Flach M, Mohle L, Rogell L, Hoyler T, Ebert K, Fabiunke C, Pfeifer D, Sexl V, Fonseca-Pereira D, Domingues R G, Veiga-Fernandes H, Arnold SJ, Busslinger M, Dunay I R, Tanriver Y, and Diefenbach A (2014). Differentiation of type 1 ILCs from a common progenitor to all helper-like innate lymphoid cell lineages. Cell 157, 340-356

  • Principal Investigator

     

     

     
     

    Prof. Dr. med Philipp Henneke

    Center of Chronic Immunodeficiencyat Center for Translational Research

    Medical Center- University of Freiburg

    Breisacher Straße 115

    79106 Freiburg

    Email: philipp.hennekeuniklinik-freiburg.de