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232. Generalized Entropy Based Clonal Diversity Estimation of TCR Repertoire
Cell Therapies I decreased platelet infusion rate and platelet count recovery after chemotherapy. This study was registered at http://www.chictr.org as # ChiCTR-TRC-11001691.
Cell Therapies I 234. Efficient Generation of Stable Genetically Modified Human iPSC-Derived Macrophages for Innovative Gene and Cell Therapeutic Strategies
232. Generalized Entropy Based Clonal Diversity Estimation of TCR Repertoire
Saira Afzal, Shahzad Ahmad, Richard Gabriel, Eliana Ruggiero, Christof von Kalle, Manfred Schmidt, Raffaele Fronza DKFZ (German Cancer Research Center), Heidelberg, Germany In depth study of T-cell receptor (TCR) repertoire, specifically clonal expansion of T cells and diversity of repertoire, can provide invaluable information in various health and diseases states including infection, cancer and genetic disorders etc. Currently, Shannon and Simpson diversity indices used to estimate TCR clonality have certain limitations. These indices are biased either towards total number of different TCR receptor types (richness) or distribution of each clonal type (evenness). We introduced here a new framework for accurate and precise quantification of clonal diversity of TCR cell population that overcomes shortcomings of already available indices. Our concept is based on the generalized form of entropy known as Rényi numbers and two components of diversity, richness and evenness. Based on the Rényi equation, we can prove that at a specific richness value the highest possible evenness obtained cannot go beyond the stated richness value. Plotting richness versus evenness helps to define maximal theoretical polyclonality and monoclonality regions and diversity of sample can be measured by estimating distances of sample from these theoretical bounds. We have validated our method on in-silico data sets and TCR repertoire study. We conclude that our idea of clonal plane and diversity index measurement provides more reliable and robust estimation of immune population diversity. It can prove to be a useful tool for quantitative characterization of TCR repertoire in immunological studies.
233. The Efficacy of Megakaryocytic Progenitor Products Generated from Cord Blood in Acute Myeloid Leukemia Patients: A Before-After Phase 2 Trial Xuetao Pei Beijing Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing, China
Infusion of cord blood-derived megakaryocytic progenitors (MPs) products shows safe and feasible for treatment of thrombocytopenia in hematological malignancies in our phase 1 study. This phase 2, beforeafter study assigned 54 patients with acute myeloid leukemia (AML) to receiving MPs products or not after consolidation chemotherapy with high-dose ara-C (HDAC). One patient from MPs group and one from control group withdraw the study. Compared to control group, the MPs group achieved a decreased platelet infusion rate (7.5% [4/53] vs.22.6% [12/53], p=0.03), platelet infusion units (1.0 vs. 2.0 Units, p=0.001), time of platelet count recovery normal (>100×109/L, p=0.01). Only two patients had reversible fever (below 38℃) and one patient had reversible local skin rash. No patients withdraw due to adverse event. MPs products were active and well tolerated, with Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Mania Ackermann1, Uta Müller-Kuller2, Sylvia Merkert3, Ulrich Martin3, Manuel Grez2, Axel Schambach4, Thomas Moritz5, Nico Lachmann1 1 JRG Translational Hematology of Congenital Diseases, Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover, Germany, 2Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany, 3 LEBAO, Department of Cardiac, Thoracic, Transplant and Vascular Surgery, REBIRTH, Hannover Medical School, Hannover, Germany, 4Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany, 5RG Reprogramming and Gene Therapy, Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover, Germany Hematopoietic differentiation of human induced pluripotent stem cells (iPSC) holds great promise for disease modeling, drug testing, and in particular for the development of novel gene and cell therapeutic strategies. In the past, interest has been directed primarily at the generation of reconstituting hematopoietic stem cells, a cell type as of yet problematic to obtain from hPSCs. Recently however, also long-lived mature myeloid cells such as tissue resident macrophages have been described and further characterized. Therefore, transplantation of macrophages may serve as an innovative treatment approach in several diseases such as pulmonary alveolar proteinosis (PAP) and adenosine deaminase deficiency (ADA). In this line, we have established an efficient, embryoid body-based human iPSC-differentiation protocol employing IL-3 in combination with M-SCF to continuously (up to 6 months) generate large numbers (5-10x106/week/6 well plate) of >95% pure monocyte/macrophages (iPSC-MΦ) via an intermediate “myeloid cell forming complex”. The iPSC-MΦ revealed typical morphology, surface phenotype and functionality comparable to their in vivo derived counterparts. In order to evaluate the suitability of this protocol also for gene therapy, we transduced human iPSC with 3rd generation SIN lentiviral vectors employing a minimal and safety optimized ubiquitous chromatin opening element (CBX3-UCOE) to prevent differentiation-associated epigenetic silencing. Here, GFP reporter gene expression driven by the elongation factor 1α short (EFS) promoter resulted in >90% GFP+ iPSC-MΦ after myeloid differentiation. In contrast, the EFS promoter without the CBX3-UCOE was subject to massive epigenetic silencing. Moreover, also a myeloid-biased myeloid-related protein 8 (Mrp8) promoter was efficiently protected from silencing, whilst tissue specificity was not affected by the CBX3-UCOE. Of note, the protective activity of the CBX3-UCOE was associated with decreased levels of repressive histone marks (H3K27me3, H3K9me3) and increased levels of active marks (phosphorylated polymerase). Alternatively, we also targeted an expression cassette to the AAVS1 locus employing zinc finger nuclease mediated homologous recombination. Selected clones showed targeted and robust CMV early enhancer/chicken β actin (CAG) promoter-driven fluorochrome expression in the pluripotent status as well as in terminally differentiated iPSC-MΦ. In summary, we here describe silencing resistance lentiviral vectors and genome editing strategies as well as a differentiation protocol allowing for the efficient and long-term generation of stable genetically modified iPSC-MΦ, thus paving the way for novel gene and cell based therapies. S91
Cell Therapies I 234. Efficient Generation of Stable Genetically Modified Human iPSC-Derived Macrophages for Innovative Gene and Cell Therapeutic Strategies
232. Generalized Entropy Based Clonal Diversity Estimation of TCR Repertoire
Saira Afzal, Shahzad Ahmad, Richard Gabriel, Eliana Ruggiero, Christof von Kalle, Manfred Schmidt, Raffaele Fronza DKFZ (German Cancer Research Center), Heidelberg, Germany In depth study of T-cell receptor (TCR) repertoire, specifically clonal expansion of T cells and diversity of repertoire, can provide invaluable information in various health and diseases states including infection, cancer and genetic disorders etc. Currently, Shannon and Simpson diversity indices used to estimate TCR clonality have certain limitations. These indices are biased either towards total number of different TCR receptor types (richness) or distribution of each clonal type (evenness). We introduced here a new framework for accurate and precise quantification of clonal diversity of TCR cell population that overcomes shortcomings of already available indices. Our concept is based on the generalized form of entropy known as Rényi numbers and two components of diversity, richness and evenness. Based on the Rényi equation, we can prove that at a specific richness value the highest possible evenness obtained cannot go beyond the stated richness value. Plotting richness versus evenness helps to define maximal theoretical polyclonality and monoclonality regions and diversity of sample can be measured by estimating distances of sample from these theoretical bounds. We have validated our method on in-silico data sets and TCR repertoire study. We conclude that our idea of clonal plane and diversity index measurement provides more reliable and robust estimation of immune population diversity. It can prove to be a useful tool for quantitative characterization of TCR repertoire in immunological studies.
233. The Efficacy of Megakaryocytic Progenitor Products Generated from Cord Blood in Acute Myeloid Leukemia Patients: A Before-After Phase 2 Trial Xuetao Pei Beijing Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing, China
Infusion of cord blood-derived megakaryocytic progenitors (MPs) products shows safe and feasible for treatment of thrombocytopenia in hematological malignancies in our phase 1 study. This phase 2, beforeafter study assigned 54 patients with acute myeloid leukemia (AML) to receiving MPs products or not after consolidation chemotherapy with high-dose ara-C (HDAC). One patient from MPs group and one from control group withdraw the study. Compared to control group, the MPs group achieved a decreased platelet infusion rate (7.5% [4/53] vs.22.6% [12/53], p=0.03), platelet infusion units (1.0 vs. 2.0 Units, p=0.001), time of platelet count recovery normal (>100×109/L, p=0.01). Only two patients had reversible fever (below 38℃) and one patient had reversible local skin rash. No patients withdraw due to adverse event. MPs products were active and well tolerated, with Molecular Therapy Volume 24, Supplement 1, May 2016 Copyright © The American Society of Gene & Cell Therapy
Mania Ackermann1, Uta Müller-Kuller2, Sylvia Merkert3, Ulrich Martin3, Manuel Grez2, Axel Schambach4, Thomas Moritz5, Nico Lachmann1 1 JRG Translational Hematology of Congenital Diseases, Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover, Germany, 2Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt, Germany, 3 LEBAO, Department of Cardiac, Thoracic, Transplant and Vascular Surgery, REBIRTH, Hannover Medical School, Hannover, Germany, 4Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany, 5RG Reprogramming and Gene Therapy, Institute of Experimental Hematology, REBIRTH, Hannover Medical School, Hannover, Germany Hematopoietic differentiation of human induced pluripotent stem cells (iPSC) holds great promise for disease modeling, drug testing, and in particular for the development of novel gene and cell therapeutic strategies. In the past, interest has been directed primarily at the generation of reconstituting hematopoietic stem cells, a cell type as of yet problematic to obtain from hPSCs. Recently however, also long-lived mature myeloid cells such as tissue resident macrophages have been described and further characterized. Therefore, transplantation of macrophages may serve as an innovative treatment approach in several diseases such as pulmonary alveolar proteinosis (PAP) and adenosine deaminase deficiency (ADA). In this line, we have established an efficient, embryoid body-based human iPSC-differentiation protocol employing IL-3 in combination with M-SCF to continuously (up to 6 months) generate large numbers (5-10x106/week/6 well plate) of >95% pure monocyte/macrophages (iPSC-MΦ) via an intermediate “myeloid cell forming complex”. The iPSC-MΦ revealed typical morphology, surface phenotype and functionality comparable to their in vivo derived counterparts. In order to evaluate the suitability of this protocol also for gene therapy, we transduced human iPSC with 3rd generation SIN lentiviral vectors employing a minimal and safety optimized ubiquitous chromatin opening element (CBX3-UCOE) to prevent differentiation-associated epigenetic silencing. Here, GFP reporter gene expression driven by the elongation factor 1α short (EFS) promoter resulted in >90% GFP+ iPSC-MΦ after myeloid differentiation. In contrast, the EFS promoter without the CBX3-UCOE was subject to massive epigenetic silencing. Moreover, also a myeloid-biased myeloid-related protein 8 (Mrp8) promoter was efficiently protected from silencing, whilst tissue specificity was not affected by the CBX3-UCOE. Of note, the protective activity of the CBX3-UCOE was associated with decreased levels of repressive histone marks (H3K27me3, H3K9me3) and increased levels of active marks (phosphorylated polymerase). Alternatively, we also targeted an expression cassette to the AAVS1 locus employing zinc finger nuclease mediated homologous recombination. Selected clones showed targeted and robust CMV early enhancer/chicken β actin (CAG) promoter-driven fluorochrome expression in the pluripotent status as well as in terminally differentiated iPSC-MΦ. In summary, we here describe silencing resistance lentiviral vectors and genome editing strategies as well as a differentiation protocol allowing for the efficient and long-term generation of stable genetically modified iPSC-MΦ, thus paving the way for novel gene and cell based therapies. S91