iPS based Haematopoietic Regeneration

Objectives

Projects of the group combine work on pluripotent stem cells (PSCs) and hematopoietic stem cells (HSCs). The PSC-part is based on the work of 2012 Nobel Laureate Shinya Yamanaka, who introduced induced pluripotent stem cells (iPSCs) as a novel and increasingly important tool to modern biomedical research. IPSCs which can easily be generated from various somatic cell sources together with their differentiated progeny qualify as powerful cellular disease models but also represent a highly promising source for cell and gene therapy. In this context, our group works on the reprogramming of somatic cells from mice and humans but also pigs and cattle (the latter two in cooperation with Prof. H. Niemann’s group at Mariensee) focusing on hematopoietic cells as a readily available target population. Major topics include the use of miRNAs to optimize reprogramming processes, the suitability of ubiquitous chromatin opening elements (UCOE) to stabilize transgene expression in PSCs and their progeny, and the development of iPSC-based gene therapy strategies for diseases caused by malfunctions of hematopoietic cells such as Pulmonary Alveolar Proteinosis (PAP). This work is performed in close cooperation with our main Rebirth Cooperation partners Prof. Dr.T. Cantz (RG Translational Hepatology and Stem Cell Biology), Prof. Dr. A. Schambach,PhD and PD Dr. Nico Lachmann (RG Translation Hematology of Congenital  Diseases) as well as Prof. Dr. U. Martin (LEBAO). With respect to HSCs and more differentiated hematopoietic cells (also see unit 6.1) interests of our group primarily involve HSC gene therapy and HSC stem cell biology. While a proof-of-principle for the feasibility of HSC gene therapy was established as early as the mid-1980s, it took the current millennium to establish clinically successful gene therapy studies for various immunodeficiency syndromes and more recently also x-linked adrenoleukodystrophy (for review see: Kohn, Clin Immunol, 2010; Cartier, Brain Pathol, 2010). Along this line, our group aims to develop efficient hematopoietic cell based gene therapy approaches for other disease states caused by hematopoietic cells malfunction or deficiency such as Pulmonary Alveolar Proteinosis (PAP) or anti-cancer chemotherapy-induced myelosuppression. Furthermore, we evaluate the use of humanized mouse models to assess the efficacy as well as the safety (genotoxicity) of hematopoietic cell gene therapy approaches. In terms of HSC stem cell biology, our projects include the ex vivo generation of primitive and differentiated hematopoietic cells from ESCs/iPSCs, a systematic analysis of the role of miRNAs in primitive reconstituting HSCs, but also the generation of suitable “humanized“ disease models to analyze and compare normal and leukemic stem cells.

Research Focus

A screening assay allowing for the identification of novel small interfering RNAs modulating iPSC generation
MicroRNA mediated effects during iPSC generation

Mechanisms of Transcription Factor-Mediated Reprogramming
As of yet, the molecular mechanisms of transcription factor-mediated reprogramming are not fully understood. In consequence, iPSC generation still is a very slow and inefficient process. Moreover, there is a great functional heterogeneity among established iPSC lines including highly variable differentiation propensities into specific cellular lineages which complicates the use of iPSCs in translational settings. Thus, we contribute to studies elucidating the mechanisms of transcription factor-mediated reprogramming in more detail in order to enhance the generation of bona fide iPSCs. These studies focus on the role of microRNAs (miRNAs), short non-coding RNAs, which have repeatedly been demonstrated to control important cell fate decisions through inhibition of translation or degradation of target mRNAs. Several miRNAs such as the miR-302 and miR-290 clusters have been identified to regulate self-renewal and differentiation processes in embryonic stem cells. Thus in cooperation with we have utilized a screening platform that allows simultaneous analysis of hundreds of miRNAs for their effect on the generation of iPSCs. These studies revealed a miRNA family consisting of miR-130b, miR-301b, and miR-721, which enhance reprogramming through inhibition of the homebox transcription factor Meox2. More recently, we also investigated miRNAs inhibitory to the generation of iPSCs and identified two mRNAs, miR-132 and mi-212 (Pfaff etal., Stem Cell Res., 2017) acting through inhibition of crucial epigenetic modifiers.

 

 

Structure of the A2UCOE and example of UCOE-containing lentiviral vectors for stabilization of transgene expression
Targeted integration into the hAAVS1 locus utilizing ZFN-mediated homologous recombination (HR).

Transgene expression in pluripotent stem cells (PSCs) and their differentiated progeny 

Ubiquitous chromatin opening elements (UCOEs): While PSC represent a promising target population for gene therapy approaches, epigenetic transgene silencing especially during PSC-differentiation constitutes a major obstacle for this strategy. Thus, we evaluate different strategies to stabilize transgene expression in PSCs. One approach is the use of ubiquitous chromatin opening element (UCOE). UCOEs are genomic regions containing two divergently transcribed promoter elements (Fig. 3.3.1) and have important chromatin remodeling functions. UCOEs have been shown to prevent silencing of retroviral transgene expression in cell lines (Antoniou et al., Genomics, 2003), HSCs (Zhang et al., Blood, 2010), and during hematopoietic differentiation of human and murine PSCs (Pfaff, et al., Stem Cells, 2013). We currently investigate UCOEs as a generalized tool to stabilize transgene expression in PSCs and thereof derived tissues. In addition, we try to decipher the molecular mechanism underlying the transgene stabilizing effect of UCOEs. Designer Nucleases: In contrast to the semi-random integration of retro/lentiviral vectors, the ideal gene therapy technology would allow site-specific editing of the human genome to direct transgenes specifically into genomic areas regarded as “safe harbors” such as the AAVS1 locus or even to enable direct “on-site” repair of a defective gene locus. While this is principally possible by homologous recombination (HR), HR-events are very rare in mammalian cells, but can be 100- to 10.000-fold stimulated, however, by introduction of a site-specific DNA double strand break utilizing designer nucleases such as zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs). Here our group in cooperation with Prof. Dr. phil. Toni Cathomen (Freiburg) evaluates the repair of the CSF2RA and CSF2RB locus (see 3.4) or the introduction of drug resistance genes into the AAVS1 site (see 3.5) as a strategy to stabilize transgene expression and minimize the risk of insertional mutagenesis (Rahman et al., Human Gene Therapy, 2011). 

Characterization of CSF2RA deficient PAP iPSCs
Scheme of iPSC-based gene therapy

Hematopoietic cell and iPSC-based gene therapy of congenital diseases of the blood and immune system

Over the last years pluripotent cells and in particular iPSCs have emerged as an increasingly important tool for gene therapy approaches. In this context, our efforts focus specifically on hereditary Pulmonary Alveolar Proteinosis (PAP), a severe congenital lung disease caused by defects in alveolar macrophage maturation and function. The hallmark of the disease is the massive protein and phospholipid accumulation in the lungs leading to progressive respiratory failure and a high susceptibility for pulmonary infections. Congenital forms of PAP are rare and result from homozygous or compound heterozygous mutations in the GM-CSF receptor genes CSF2RA or CSF2RB encoding the a- and ß-subunit of the GM-CSF receptor. Repetitive whole lung lavage associated with many hours of general anesthesia and mechanical ventilation, constitutes the only effective treatment of CSF2RA-/--PAP, and both, quality of life and life expectancy are significantly reduced. Hence, novel therapeutic strategies for this disease are highly desirable. Our group has established the organotropic (pulmonary) delivery of monocyte/macrophages generated ex vivo from genetically repaired PAP-patient-specific CD34+ hematopoietic cells or iPSCs as an innovative and cause-oriented cell and gene therapy strategy for PAP. Furthermore, given that HSCs life-long give rise to all the different hematopoietic cells of an individual, HSCs represent one of the most attractive target cells of gene therapy approaches. So far, most of these activities have addressed immunodeficiencies, but we currently also explore HSCs or thereof derived differentiated cells such as macrophages as cell sources for gene therapy strategies in PAP or other congenital diseases.

Scheme of HSC-based gene therapy with drug resistance (CTX-R) genes.
Regulation of transgene expression by doxycycline-induced (Tet-on) and miRNA-mediated expression systems

HSC-based gene therapy approaches for myeloprotection

Although HSC gene therapy so far primarily has been applied to the treatment of congenital diseases, another potentially attractive application is the transfer of drug resistance (CTX-R) genes to protect the lympho-hematopoietic system from the side effects of anti-cancer chemotherapy, an approach referred to as chemo- or myeloprotective gene therapy (Moritz et al, Cytotherapy, 2001). In addition, expression of CTX-R genes can provide an effective selective system allowing for in vitro and in vivo enrichment of gene-modified HSCs by drug application. A number of CTX-R genes have been identified which protect against many of the clinically important anti-cancer agents, and for the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT), the context of myeloprotection recently has been clinically verified in the context of glioblastoma chemotherapy (Adair et al., Science Trans Med, 2012). Two other clinically relevant CTX-R genes are cytidine deaminase (CDD) protecting cells from cytidine-analogue-type drugs such as cytosine arabinoside (Ara-C) and the multidrug resistance gene 1 (MDR1) protecting against a wide variety of agents including anthracyclines, taxoids, epidophyllotoxins or vinca-alkaloids. Lentiviral-mediated expression of CDD and MDR1 will be explored in relevant murine disease models of leukemia and myelodysplastic/myeloproliferative diseases to develop novel and safety-improved treatment strategies for these disease entities. Furthermore, we employ regulated CTX-R gene expression systems including inducible (Tet-ON) and microRNA-regulated lentiviral vectors for transgene expression. Finally, we are interested in identifying and utilizing new drug resistance mechanisms in the context of HSC gene therapy such as knock-down of the deoxycytidine kinase (dCK) represent the most promising candidates.

 

 

 

Humanized’ mouse model to assess the genotoxicity of viral vectors in the context of MGMT-mediated in vivo selection.

Genotoxicity assessment utilizing humanized mouse model

Despite the considerable success of recent years, clinical studies on HSC gene therapy also have demonstrated how integrating vectors can impede the field. Thus, viral vectors may lead to activation of proto-oncogenes or disrupt cellular gene function resulting into a proliferative advantage of affected cells giving rise to clonal dominance or even overt leukemias (Rivat et al., Hum Gene Ther, 2012). At present, preclinical genotoxicity assessment is performed in murine in vitro immortalization or in vivo transplant assays, which have been validated for this purpose (Modlich et al. Blood, 2006 & Leukemia, 2008). However, due to species differences direct investigation of the genetically modified human cell product in relevant “humanized” mouse models based on immunodeficient mouse strains (Shultz et al., Nat Rev Immunol, 2007) appears desirable. To evaluate the feasibility of humanized mice based preclinical assay systems for the assessment of genotoxic events we currently investigate two approaches characterized by considerable risk for clonal selection of retrovirally transduced human HSCs: (i) MGMT-based in vivo selection of genetically modified HSCs and (ii) prolonged ex vitro HSC expansion using different cytokine combinations. To gain an insight into the clonal repertoire of human reconstitution in these models, we perform integration analysis using ligation mediated PCR followed by high throughput sequencing on peripheral blood and bone marrow samples. 

 

 

Collaborations

  • B. Trapnell, Cincinnati Children’s: Gene Therapy of Hereditary Pulmonary Alveolar Proteinosis (herPAP) based on Pulmonary Macrophage Transfer (PMT).
  • T. Cantz, REBIRTH Unit Translational Hepatology and Stem Cell Biology: Transgene expression during endodermal differentiation, role of miRNAs in reprogramming, shared platforms for iPSC-generation, joint lab meetings, and common protocols to facilitate exchange of expertise and intergroup cooperations. 
  • A. Schambach, B. Schiedlmeier, M. Galla, J. Bode, Institute of Experimental Hematology, MHH: Lentiviral vectors for effective and safety-improved reprogramming, protocols and models for HSC expansion, ESC/iPSC-based generation of hematopoietic cells, HSC- and iPSC-based gene therapy strategies. 
  • U. Martin, REBIRTH Unit iPSs for Disease Modelling, Drug Screening and Cell Therapy, R. Zweigerdt REBIRTH Unit Mass Production of Pluripotent Stem Cells and Derivates, I. Gruh, REBIRTH Unit Myocardial Tissue Engineering, LEBAO, MHH: Role of “UCOE “ elements in iPSC-based gene therapy of cardiac diseases, iPSC-based gene therapy of congenital diseases, visualisation of iPSC differentiation and gene transfer of fluorescence markers 
  • N. Lachmann, REBIRTH Unit Translational Hematology of Congenital Diseases 
  • H. Niemann & W. Kues (ING, FLI Mariensee): Generation and characterization of porcine and bovine iPSCs. Analysis of porcine iPS derived therapeutic cells in syngenic large animal model derived by somatic cell nuclear transfer (SCNT). 
  • A. Deep Sharma, REBIRTH Unit MiRNA in liver regeneration: Role of miRNA in hematopoietic differentiation, Transgene expression during endodermal differentiation 
  • B. Schlegelberger, D. Steinemann, REBIRTH Unit Genomic Profiling & G. Göhring, REBIRTH Unit Cytogenetic Profiling and , MHH: Chromosomal analysis of iPSCs 
  • R. Stripecke, REBIRTH Unit Regenerative Immune Therapies, MHH: Immune response to iPSCs and iPSC-derived cells 
  • Th. Thum, REBIRTH Unit miRNA in Myocardial Regeneration, IMTTS, MHH: Role of miRNAs in reprogramming and early hematopoietic differentiation 
  • G. Hansen, C. Happle, MHH & B. Trapnell, Cincinnati: Generation of disease-specific iPSCs from PAP-patients & Gene Therapy of Pulmonary Alvealar Proteinosis (PAP) 
  • U. Modlich, Frankfurt: iPSC–based therapy of mpl-deficiency & safety analysis of HSC gene transfer.
  • T. Cathomen, Freiburg: Use of ZF- and TALE-nucleases for iPSC-based gene therapy approaches 
  • J. Thomale, Essen & H. Geiger, Ulm: Analysis of genetic lesions and their repair in HSCs and iPSCs 
  • F. Bengel, REBIRTH Unit 8.3 Radionuclide Molecular Imaging, & J. Bankstahl, MHH: Non invasive in vivo trecking of genetically modified cells
  • D. Dilloo, Bonn: iPSC generation from leukemias and other malignancie
  • M. Heuser, MHH: Animal models and gene therapy approaches for acute leukemias
  • Ch. Buchholz, Langen: Surface-pseudotyped vectors for transduction of hematopoietic cells
  • S. Cowley, Oxford: Hematopoietic differentiation of human iPSCs
  • M. Grez, Frankfurt & M. Antoniou, London: Role of ubiquitous opening elements in iPSC/ESC and their progeny
  • D. Williams, Boston: Gene Transfer of DNA repair proteins to HSCs, iPSC-based gene therapy, role of rho-GTPases in CLL.

Current Research Funding

  • BMBF,“Genetically corrected iPSC derived macrophages (i-MAC) for innovative gene therapy strategies”, Research Alliance, Moritz, Thomas MD; coordinator , PI Subproject 4. Moritz, Thomas MD, Hansen, Gesine MD; (BMBF 01EK1602A) 3/2017-2/2020
  • DFG, „In-vitro Generierung von modifizierten Thrombozyten mit erweiterter Funktionalität aus induziert pluripotenten Stammzellen“ PIs: T. Moritz & U. Modlich, (MO 886/8-1)
  • Deutsche Krebshilfe, “From CARs to TRUCKs: Induction of a concerted antitumor immune response by engineered T cells”, Research Alliance, PIs: T. Moritz et al..
  • DFG, „Verbesserte Transgenexpression in pluripotenten Stammzellen und aus ihnen abgeleiteten Geweben durch den Einsatz von Ubiquitous Chromatin Opening Elements (UCOE)“ Moritz, Thomas MD; German Academic Research Council (DFG; MO 886/6-1) 8/2013-7/2017
  • Else Kröner-Fresenius-Stiftung, „ Intracheale Transplantation gentherapeutisch korrigierter Monozyten als innovativer Therapieansatz bei der pulmonalen Alveolarproteinose“ PIs. Moritz, Thomas MD, Hansen, Gesine MD;( 10/2013-9/2017
  • DFG, „Cytidin-Deaminase als Selektionsmarker im Rahmen der Gentherapie hämatologischer Erkrankungen“, T. Moritz, MO 886-4.
  • DFG, “Humanized models to access the genotoxicity of viral vectors in the context of hematopoietic cell expansion and in vivo selection“, PIs: T. Moritz & U. Modlich (IEH, MHH) Priority Program SPP 1230, MO 884-3.
  • HiFL career awards for several members

Awards

  • “Eva Luise Köhler Research Award for Rare Diseases 2013” dedicated to Prof. Gesine Hansen, Dr. Christine Happle, Dr. Nico Lachmann and Prof. Thomas Moritz, February 2013, for the project “Innovative Gentherapie bei seltenen monogenen Erkrankungen der Lunge”
  • Excellence in Research Award (2016) to A. Mucci, American Society for Gene and Cell Therapy (ASGCT); Washington D.C., USA
  • Top Abstract Award & Best Translational Research (2014),  to N. Lachmann, Deutsche Gesellschaft für Hämatologie und Onkologie (DGHO), Hamburg, Germany
  • Top Abstract Award and Presidential Symposium presentation, (2014) to N. Lachmann, American Society for Gene and Cell Therapy (ASGCT); Washington D.C., USA
  • “The Berrie Hesp Scholarship” (2013) to N. Pfaff, Keystone Symposium “Noncoding RNAs in Development and Cancer”, Vancouver. Canada

Publications

2013 - ongoing

2018

Happle C*, Lachmann N*, Ackermann M, Mirenska A, Göhring G, Thomey K, Mucci A, Hetzel M, Glomb T, Susuki T, Chalk C, Glage S, Dittrich-Breiholz O, Trapnell B, Moritz T*, Hansen G*. Pulmonary Transplantation of human iPSC-derived Macrophages ameliorates Pulmonary Alveolar Proteinosis. Am J Respir Crit Care Med 2018; (in press).*contributed equally

2017

Pfaff N, Liebhaber S, Mobus S, Beh-Pajooh A, Fiedler J, Pfanne A, Schambach A, Thumc T, Cantz T, Moritz T. Inhibition of miRNA-212/132 improves the reprogramming of fibroblasts into induced pluripotent stem cells by de-repressing important epigenetic remodelling factors. Stem Cell Research. 2017;20:70-5.

Kuhn A, Ackermann M, Mussolino C, Cathomen T, Lachmann N, Moritz T. TALEN-mediated functional correction of human iPSC-derived macrophages in context of hereditary pulmonary alveolar proteinosis. Sci Rep. 2017/11/11 ed2017. p. 15195.

Hetzel M, Suzuki T, Hashtchin AR, Arumugam P, Carey B, Schwabbauer M, Kuhn A, Meyer J, Schambach A, Van Der Loo J, Moritz T, Trapnell BC, Lachmann N. Function and Safety of Lentivirus-Mediated Gene Transfer for CSF2RA-Deficiency. Hum Gene Ther Methods. 2017/09/01 ed2017.

Kunkiel J, Godecke N, Ackermann M, Hoffmann D, Schambach A, Lachmann N, Wirth D, Moritz T. The CpG-sites of the CBX3 ubiquitous chromatin opening element are critical structural determinants for the anti-silencing function. Sci Rep. 2017/08/13 ed2017. p. 7919.

Ackermann M, Kuhn A, Kunkiel J, Merkert S, Martin U, Moritz T, Lachmann N. Ex vivo Generation of Genetically Modified Macrophages from Human Induced Pluripotent Stem Cells. Transfus Med Hemother. 2017/06/20 ed2017. p. 135-42.

2016

Mucci A, Kunkiel J, Suzuki T, Brennig S, Glage S, Kuhnel MP, Ackermann M, Happle C, Kuhn A, Schambach A, Trapnell BC, Hansen G, Moritz T, Lachmann N. Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency. Stem cell reports. 2016;7(2):292-305.

Borger AK, Eicke D, Wolf C, Gras C, Aufderbeck S, Schulze K, Engels L, Eiz-Vesper B, Schambach A, Guzman CA, Lachmann N, Moritz T, Martin U, Blasczyk R, Figueiredo C. Generation of HLA-universal iPSCs-derived megakaryocytes and platelets for survival under refractoriness conditions. Molecular medicine. 2016;22.

2015

Lachmann N, Czarnecki K, Brennig S, Phaltane R, Heise M, Heinz N, Kempf H, Dilloo D, Kaever V, Schambach A, Heuser M, Moritz T. Deoxycytidine-kinase knockdown as a novel myeloprotective strategy in the context of fludarabine, cytarabine or cladribine therapy. Leukemia. 2015;29(11):2266-9.

Lachmann N, Brennig S, Hillje R, Schermeier H, Phaltane R, Dahlmann J, Gruh I, Heinz N, Schiedlmeier B, Baum C, Moritz T. Tightly regulated 'all-in-one' lentiviral vectors for protection of human hematopoietic cells from anticancer chemotherapy. Gene therapy. 2015;22(11):883-92.

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

Brennig S, Lachmann N, Buchegger T, Hetzel M, Schambach A, Moritz T. Chemoprotection of murine hematopoietic cells by combined gene transfer of cytidine deaminase (CDD) and multidrug resistance 1 gene (MDR1). Journal of experimental & clinical cancer research : CR. 2015;34:148.

Brennig S, Lachmann N, Buchegger T, Hetzel M, Schambach A, Moritz T. Chemoprotection of Murine Hematopoietic Cells by Combined Gene Transfer of Cytidine Deaminase (Cdd) and Multidrug Resistance 1 Gene (Mdr1). Journal of Experimental & Clinical Cancer Research. 2015;34(1):1-12.

Lachmann N, Ackermann M, Frenzel E, Liebhaber S, Brennig S, Happle C, Hoffmann D, Klimenkova O, Luttge D, Buchegger T, Kuhnel MP, Schambach A, Janciauskiene S, Figueiredo C, Hansen G, Skokowa J, Moritz T. Large-Scale Hematopoietic Differentiation of Human Induced Pluripotent Stem Cells Provides Granulocytes or Macrophages for Cell Replacement Therapies. Stem Cell Reports. 2015;4(2):282-96.

Ackermann M, Liebhaber S, Klusmann JH, Lachmann N. Lost in Translation: Pluripotent Stem Cell-Derived Hematopoiesis. EMBO Mol Med. 2015;7(11):1388-402.

Lachmann N, Czarnecki K, Brennig S, Phaltane R, Heise M, Heinz N, Kempf H, Dilloo D, Kaever V, Schambach A, Heuser M, Moritz T. Deoxycytidine-Kinase Knockdown as a Novel Myeloprotective Strategy in the Context of Fludarabine, Cytarabine or Cladribine Therapy. Leukemia. 2015;29(11):2266-9. Epub 2015/04/30.

Lachmann N, Brennig S, Hillje R, Schermeier H, Phaltane R, Dahlmann J, Gruh I, Heinz N, Schiedlmeier B, Baum C, Moritz T. Tightly Regulated 'All-in-One' Lentiviral Vectors for Protection of Human Hematopoietic Cells from Anticancer Chemotherapy. Gene Ther. 2015;22(11):883-92.

Dreyer AK, Hoffmann D, Lachmann N, Ackermann M, Steinemann D, Timm B, Siler U, Reichenbach J, Grez M, Moritz T, Schambach A, Cathomen T. Talen-Mediated Functional Correction of X-Linked Chronic Granulomatous Disease in Patient-Derived Induced Pluripotent Stem Cells. Biomaterials. 2015;69:191-200.

Muller-Kuller U, Ackermann M, Kolodziej S, Brendel C, Fritsch J, Lachmann N, Kunkel H, Lausen J, Schambach A, Moritz T, Grez M. A Minimal Ubiquitous Chromatin Opening Element (Ucoe) Effectively Prevents Silencing of Juxtaposed Heterologous Promoters by Epigenetic Remodeling in Multipotent and Pluripotent Stem Cells. Nucleic Acids Res. 2015;43(3):1577-92.

2014

Haemmerle R, Phaltane R, Rothe M, Schroder S, Schambach A, Moritz T, Modlich U. Clonal Dominance with Retroviral Vector Insertions near the Angpt1 and Angpt2 Genes in a Human Xenotransplant Mouse Model. Mol Ther Nucleic Acids. 2014;3:e200. Epub 2014/10/08.

Lachmann N, Happle C, Ackermann M, Luttge 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, Gohring G, Cantz T, Martin U, Schwerk N, Hansen G, Moritz T. Gene Correction of Human Induced Pluripotent Stem Cells Repairs the Cellular Phenotype in Pulmonary Alveolar Proteinosis. Am J Respir Crit Care Med. 2014;189(2):167-82. Epub 2013/11/28.

Ackermann M, Lachmann N, Hartung S, Eggenschwiler R, Pfaff N, Happle C, Mucci A, Gohring G, Niemann H, Hansen G, Schambach A, Cantz T, Zweigerdt R, Moritz T. Promoter and Lineage Independent Anti-Silencing Activity of the A2 Ubiquitous Chromatin Opening Element for Optimized Human Pluripotent Stem Cell-Based Gene Therapy. Biomaterials. 2014;35(5):1531-42.

Phaltane R, Haemmerle R, Rothe M, Modlich U, Moritz T. Efficiency and Safety of O(6)-Methylguanine DNA Methyltransferase (Mgmt(P140k))-Mediated in Vivo Selection in a Humanized Mouse Model. Hum Gene Ther. 2014;25(2):144-55. Epub 2013/11/14.

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. Pulmonary Macrophage Transplantation Therapy. Nature. 2014;514(7523):450-4. Epub 2014/10/03.

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. Pulmonary Transplantation of Macrophage Progenitors as Effective and Long-Lasting Therapy for Hereditary Pulmonary Alveolar Proteinosis. Sci Transl Med. 2014;6(250):250ra113. Epub 2014/08/22.

Phaltane R, Lachmann N, Brennig S, Ackermann M, Modlich U, Moritz T. Lentiviral Mgmt(P140k)-Mediated in Vivo Selection Employing a Ubiquitous Chromatin Opening Element (A2ucoe) Linked to a Cellular Promoter. Biomaterials. 2014;35(25):7204-13. Epub 2014/05/31.

Lachmann N, Brennig S, Moritz T. Chapter 29 - Cytidine Deaminase in Myeloprotective Gene Therapy. In: Gerson ECLL, editor. Gene Therapy of Cancer (Third Edition). San Diego: Academic Press; 2014. p. 423-40.

2013

Kues WA, Herrmann D, Barg-Kues B, Haridoss S, Nowak-Imialek M, Buchholz T, Streeck M, Grebe A, Grabundzija I, Merkert S, Martin U, Hall VJ, Rasmussen MA, Ivics Z, Hyttel P, Niemann H. Derivation and Characterization of Sleeping Beauty Transposon-Mediated Porcine Induced Pluripotent Stem Cells. Stem Cells Dev. 2013;22(1):124-35.

Lachmann N, Brennig S, Pfaff N, Schermeier H, Dahlmann J, Phaltane R, Gruh I, Modlich U, Schambach A, Baum C, Moritz T. Efficient in Vivo Regulation of Cytidine Deaminase Expression in the Haematopoietic System Using a Doxycycline-Inducible Lentiviral Vector System. Gene Ther. 2013;20(3):298-307.

Pfaff N, Cantz T. From Skin to Blood: A New Member Joins the Iclub. Cell Stem Cell. 2013;13(2):131-3.

Lachmann N, Brennig S, Phaltane R, Flasshove M, Dilloo D, Moritz T. Myeloprotection by Cytidine Deaminase Gene Transfer in Antileukemic Therapy. Neoplasia. 2013;15(3):239-48.

Volkmann I, Kumarswamy R, Pfaff N, Fiedler J, Dangwal S, Holzmann A, Batkai S, Geffers R, Lother A, Hein L, Thum T. Microrna-Mediated Epigenetic Silencing of Sirtuin1 Contributes to Impaired Angiogenic Responses. Circ Res. 2013;113(8):997-1003.

Schambach A, Moritz T. Toward Position-Independent Retroviral Vector Expression in Pluripotent Stem Cells. Mol Ther. 2013;21(8):1474-7.

Pfaff N, Lachmann N, Ackermann M, Kohlscheen S, Brendel C, Maetzig T, Niemann H, Antoniou MN, Grez M, Schambach A, Cantz T, Moritz T. A Ubiquitous Chromatin Opening Element Prevents Transgene Silencing in Pluripotent Stem Cells and Their Differentiated Progeny. Stem Cells. 2013;31(3):488-99.

2006 - 2012

2012

Pfaff N, Lachmann N, Kohlscheen S, Sgodda M, Arauzo-Bravo MJ, Greber B, Kues W, Glage S, Baum C, Niemann H, Schambach A, Cantz T, Moritz T. Efficient Hematopoietic Redifferentiation of Induced Pluripotent Stem Cells Derived from Primitive Murine Bone Marrow Cells. Stem Cells Dev. 2012;21(5):689-701.

Pfaff N, Moritz T, Thum T, Cantz T. Mirnas Involved in the Generation, Maintenance, and Differentiation of Pluripotent Cells. J Mol Med (Berl). 2012;90(7):747-52.

Lachmann N, Jagielska J, Heckl D, Brennig S, Pfaff N, Maetzig T, Modlich U, Cantz T, Gentner B, Schambach A, Moritz T. Microrna-150-Regulated Vectors Allow Lymphocyte-Sparing Transgene Expression in Hematopoietic Gene Therapy. Gene Ther. 2012;19(9):915-24.

Brennig S, Rattmann I, Lachmann N, Schambach A, Williams DA, Moritz T. In Vivo Enrichment of Cytidine Deaminase Gene-Modified Hematopoietic Cells by Prolonged Cytosine-Arabinoside Application. Cytotherapy. 2012;14(4):451-60.

2011

Giordano FA, Sorg UR, Appelt JU, Lachmann N, Bleier S, Roeder I, Kleff V, Flasshove M, Zeller WJ, Allgayer H, von Kalle C, Fruehauf S, Moritz T, Laufs S. Clonal Inventory Screens Uncover Monoclonality Following Serial Transplantation of Mgmt P140k-Transduced Stem Cells and Dose-Intense Chemotherapy. Hum Gene Ther. 2011;22(6):697-710.

Hauschild J, Petersen B, Santiago Y, Queisser AL, Carnwath JW, Lucas-Hahn A, Zhang L, Meng X, Gregory PD, Schwinzer R, Cost GJ, Niemann H. Efficient Generation of a Biallelic Knockout in Pigs Using Zinc-Finger Nucleases. Proc Natl Acad Sci U S A. 2011;108(29):12013-7.

Garrels W, Mates L, Holler S, Dalda A, Taylor U, Petersen B, Niemann H, Izsvak Z, Ivics Z, Kues WA. Germline Transgenic Pigs by Sleeping Beauty Transposition in Porcine Zygotes and Targeted Integration in the Pig Genome. PLoS One. 2011;6(8):e23573.

Pfaff N, Fiedler J, Holzmann A, Schambach A, Moritz T, Cantz T, Thum T. Mirna Screening Reveals a New Mirna Family Stimulating Ips Cell Generation Via Regulation of Meox2. EMBO Rep. 2011;12(11):1153-9.

Nowak-Imialek M, Kues WA, Petersen B, Lucas-Hahn A, Herrmann D, Haridoss S, Oropeza M, Lemme E, Scholer HR, Carnwath JW, Niemann H. Oct4-Enhanced Green Fluorescent Protein Transgenic Pigs: A New Large Animal Model for Reprogramming Studies. Stem Cells Dev. 2011;20(9):1563-75.

Iqbal K, Kues WA, Baulain U, Garrels W, Herrmann D, Niemann H. Species-Specific Telomere Length Differences between Blastocyst Cell Compartments and Ectopic Telomere Extension in Early Bovine Embryos by Human Telomerase Reverse Transcriptase. Biol Reprod. 2011;84(4):723-33.

Aydin S, Grabellus F, Eisele L, Mollmann M, Hanoun M, Ebeling P, Moritz T, Carpinteiro A, Nuckel H, Sak A, Gothert JR, Duhrsen U, Durig J. Investigating the Role of Cd38 and Functionally Related Molecular Risk Factors in the Cll Nod/Scid Xenograft Model. Eur J Haematol. 2011;87(1):10-9.

2010

Nowak-Imialek M, Kues WA, Rudolph C, Schlegelberger B, Taylor U, Carnwath JW, Niemann H. Preferential Loss of Porcine Chromosomes in Reprogrammed Interspecies Cell Hybrids. Cell Reprogram. 2010;12(1):55-65.

Sanchez-Aguilera A, Rattmann I, Drew DZ, Muller LU, Summey V, Lucas DM, Byrd JC, Croce CM, Gu Y, Cancelas JA, Johnston P, Moritz T, Williams DA. Involvement of Rhoh Gtpase in the Development of B-Cell Chronic Lymphocytic Leukemia. Leukemia. 2010;24(1):97-104.

2009

Funke S, Schneider IC, Glaser S, Muhlebach MD, Moritz T, Cattaneo R, Cichutek K, Buchholz CJ. Pseudotyping Lentiviral Vectors with the Wild-Type Measles Virus Glycoproteins Improves Titer and Selectivity. Gene Ther. 2009;16(5):700-5.

2008

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. Gene Therapy of Beta(C)-Deficient Pulmonary Alveolar Proteinosis (Beta(C)-Pap): Studies in a Murine in Vivo Model. Mol Ther. 2008;16(4):757-64.

Kues WA, Sudheer S, Herrmann D, Carnwath JW, Havlicek V, Besenfelder U, Lehrach H, Adjaye J, Niemann H. Genome-Wide Expression Profiling Reveals Distinct Clusters of Transcriptional Regulation During Bovine Preimplantation Development in Vivo. Proc Natl Acad Sci U S A. 2008;105(50):19768-73.

Milsom MD, Jerabek-Willemsen M, Harris CE, Schambach A, Broun E, Bailey J, Jansen M, Schleimer D, Nattamai K, Wilhelm J, Watson A, Geiger H, Margison GP, Moritz T, Baum C, Thomale J, Williams DA. Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140k Expression Level and Chemoprotection of Hematopoietic Stem Cells. Cancer Res. 2008;68(15):6171-80.

Nowak-Imialek M, Wrenzycki C, Herrmann D, Lucas-Hahn A, Lagutina I, Lemme E, Lazzari G, Galli C, Niemann H. Messenger Rna Expression Patterns of Histone-Associated Genes in Bovine Preimplantation Embryos Derived from Different Origins. Mol Reprod Dev. 2008;75(5):731-43.

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