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mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5

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 CCR...

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Autores principales: Mock, Ulrike, Machowicz, Rafał, Hauber, Ilona, Horn, Stefan, Abramowski, Pierre, Berdien, Belinda, Hauber, Joachim, Fehse, Boris
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477672/
https://www.ncbi.nlm.nih.gov/pubmed/25964300
http://dx.doi.org/10.1093/nar/gkv469
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author Mock, Ulrike
Machowicz, Rafał
Hauber, Ilona
Horn, Stefan
Abramowski, Pierre
Berdien, Belinda
Hauber, Joachim
Fehse, Boris
author_facet Mock, Ulrike
Machowicz, Rafał
Hauber, Ilona
Horn, Stefan
Abramowski, Pierre
Berdien, Belinda
Hauber, Joachim
Fehse, Boris
author_sort Mock, Ulrike
collection PubMed
description 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-1(BaL) strain. Long-term exposure to HIV-1(BaL) 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.
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spelling pubmed-44776722015-06-29 mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5 Mock, Ulrike Machowicz, Rafał Hauber, Ilona Horn, Stefan Abramowski, Pierre Berdien, Belinda Hauber, Joachim Fehse, Boris Nucleic Acids Res Nucleic Acid Enzymes 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-1(BaL) strain. Long-term exposure to HIV-1(BaL) 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. Oxford University Press 2015-06-23 2015-05-11 /pmc/articles/PMC4477672/ /pubmed/25964300 http://dx.doi.org/10.1093/nar/gkv469 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Nucleic Acid Enzymes
Mock, Ulrike
Machowicz, Rafał
Hauber, Ilona
Horn, Stefan
Abramowski, Pierre
Berdien, Belinda
Hauber, Joachim
Fehse, Boris
mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title_full mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title_fullStr mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title_full_unstemmed mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title_short mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5
title_sort mrna transfection of a novel tal effector nuclease (talen) facilitates efficient knockout of hiv co-receptor ccr5
topic Nucleic Acid Enzymes
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477672/
https://www.ncbi.nlm.nih.gov/pubmed/25964300
http://dx.doi.org/10.1093/nar/gkv469
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