title: Generation of Genetically Engineered Precursor T-Cells From Human Umbilical Cord Blood Using an Optimized Alpharetroviral Vector Platform.
abstract: Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.|Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany.
authors.forename: Juwita
authors.initials: J
authors.lastname: Hübner
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
authors.forename: Shahabuddin S
authors.initials: SS
authors.lastname: Hoseini
authors.affiliation: Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Julia D
authors.initials: JD
authors.lastname: Suerth
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Dirk
authors.initials: D
authors.lastname: Hoffmann
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
authors.forename: Marcel
authors.initials: M
authors.lastname: Maluski
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Herbst
authors.affiliation: Department of Gynecology and Obstetrics, Marienkrankenhaus, Hamburg, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Maul
authors.affiliation: Department of Immunology and Medicine, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: Arnab
authors.initials: A
authors.lastname: Ghosh
authors.affiliation: Institute for Transfusion Medicine, Hannover Medical School, Hannover, Germany.
authors.forename: Britta
authors.initials: B
authors.lastname: Eiz-Vesper
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Qinggong
authors.initials: Q
authors.lastname: Yuan
authors.affiliation: Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Ott
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany.|Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Heuser
authors.affiliation: Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany.|Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Schambach
authors.affiliation: Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.|Integrated Research and Treatment Center Transplantation (IFB-Tx), Hannover Medical School, Hannover, Germany.
authors.forename: Martin G
authors.initials: MG
authors.lastname: Sauer
mesh_terms: D001355:Alpharetrovirus / Q000235:genetics*; D018952:Antigens, CD34 / Q000378:metabolism; D017209:Apoptosis; D001425:Bacterial Outer Membrane Proteins; D015415:Biomarkers; D002454:Cell Differentiation; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005818:Genetic Engineering; D005822:Genetic Vectors / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology; D010641:Phenotype; D054504:Precursor Cells, T-Lymphoid / Q000166:cytology* / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: D018952:Antigens, CD34; D001425:Bacterial Outer Membrane Proteins; D015415:Biomarkers; D053650:Interleukin-3 Receptor alpha Subunit; D011948:Receptors, Antigen, T-Cell; C418865:membrane protein, 75kDa