title: mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.
abstract: Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Mock
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany Department of Hematology, Oncology and Internal Medicine, Medical University of Warsaw, 02-097, Poland.
authors.forename: Rafał
authors.initials: R
authors.lastname: Machowicz
authors.affiliation: Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, 20251, Germany.
authors.forename: Ilona
authors.initials: I
authors.lastname: Hauber
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Horn
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany.
authors.forename: Pierre
authors.initials: P
authors.lastname: Abramowski
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany.
authors.forename: Belinda
authors.initials: B
authors.lastname: Berdien
authors.affiliation: Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, 20251, Germany German Center for Infection Research (DZIF), partner site Hamburg, Hamburg, Germany.
authors.forename: Joachim
authors.initials: J
authors.lastname: Hauber
authors.affiliation: Research Dept. Cell and Gene Therapy, Dept. of Stem Cell Transplantation, University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, 20246, Germany fehse@uke.de.
authors.forename: Boris
authors.initials: B
authors.lastname: Fehse
mesh_terms: D002460:Cell Line; D059766:DNA End-Joining Repair; D003851:Deoxyribonucleases / Q000737:chemistry / Q000235:genetics*; D018274:Electroporation; D055786:Gene Knockout Techniques / Q000379:methods*; D006678:HIV / Q000502:physiology; D015658:HIV Infections / Q000235:genetics; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger; D019713:Receptors, CCR5 / Q000235:genetics*; D013601:T-Lymphocytes / Q000821:virology; D014162:Transfection
chemical_list: D012333:RNA, Messenger; D019713:Receptors, CCR5; D003851:Deoxyribonucleases