title: TALEN-mediated editing of endogenous T-cell receptors facilitates efficient reprogramming of T lymphocytes by lentiviral gene transfer.
abstract: Adoptive immunotherapy with T lymphocytes expressing transgenic T-cell receptors (TCRs) has shown significant clinical efficacy in various malignant diseases. However, concurrent expression of endogenous and transgenic TCRs in one and the same T cell may impair efficacy and cause safety problems owing to mispairings. The most elegant approach to address these issues is the complete shutoff of the endogenous receptor chains by genome editing. To this end, we designed TCR-α and TCR-β-specific pairs of transcription activator-like effector nucleases (TALENs). TALENs were delivered into T cells using an optimized messenger RNA-electroporation protocol. Based thereon, we obtained precise and highly efficient knockout (KO) in Jurkat (TCR-α: 59.7 ± 4.0%, TCR-β: 37.4 ± 7.3%) as well as primary T cells (TCR-α: 58.0 ± 15.0%, TCR-β: 41.0 ± 17.6%). Moreover, a successive KO strategy for the endogenous TCR chains combined with subsequent transduction of the respective chains of an Influenza virus-specific model TCR led to complete reprogramming of T cells with strongly improved expression and functionality of transgenic TCRs. In conclusion, we have developed novel means for the efficient genome editing in primary T lymphocytes.
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, UCCH, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Berdien
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, UCCH, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Mock
authors.affiliation: Department of Oncology/Hematology, UMC Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Atanackovic
authors.affiliation: Research Department Cell and Gene Therapy, Department of Stem Cell Transplantation, UCCH, University Medical Center (UMC) Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: B
authors.initials: B
authors.lastname: Fehse
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics; D018274:Electroporation / Q000379:methods; D055786:Gene Knockout Techniques; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D019169:Jurkat Cells; D016086:Lentivirus / Q000235:genetics*; D010957:Plasmids; D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013601:T-Lymphocytes / Q000502:physiology*; D014162:Transfection / Q000379:methods
chemical_list: D012333:RNA, Messenger; D016693:Receptors, Antigen, T-Cell, alpha-beta; D011994:Recombinant Proteins; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific