Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

Yujia Cai; Anders Laustsen; Yan Zhou; Chenglong Sun; Mads Valdemar Anderson; Shengting Li; Niels Uldbjerg; Yonglun Luo; Martin R. Jakobsen; Jacob Giehm Mikkelsen

doi:10.7554/eLife.12213

Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

Yujia Cai, Anders Laustsen, Yan Zhou, Chenglong Sun, Mads Valdemar Anderson, Shengting Li, Niels Uldbjerg, Yonglun Luo, Martin R. Jakobsen, Jacob Giehm Mikkelsen

Novo Nordisk Foundation Center for Biosustainability

Aarhus University

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Abstract

Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et et, 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZEN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34⁺ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies.

Original language	English
Article number	e12213
Journal	eLife
Volume	5
Number of pages	15
ISSN	2050-084X
DOIs	https://doi.org/10.7554/eLife.12213
Publication status	Published - 2016

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This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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title = "Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors",

abstract = "Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et et, 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZEN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies.",

author = "Yujia Cai and Anders Laustsen and Yan Zhou and Chenglong Sun and Anderson, {Mads Valdemar} and Shengting Li and Niels Uldbjerg and Yonglun Luo and Jakobsen, {Martin R.} and {Giehm Mikkelsen}, Jacob",

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T1 - Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

AU - Cai, Yujia

AU - Laustsen, Anders

AU - Zhou, Yan

AU - Sun, Chenglong

AU - Anderson, Mads Valdemar

AU - Li, Shengting

AU - Uldbjerg, Niels

AU - Luo, Yonglun

AU - Jakobsen, Martin R.

AU - Giehm Mikkelsen, Jacob

N1 - This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

PY - 2016

Y1 - 2016

N2 - Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et et, 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZEN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies.

AB - Biased integration remains a key challenge for gene therapy based on lentiviral vector technologies. Engineering of next-generation lentiviral vectors targeting safe genomic harbors for insertion is therefore of high relevance. In a previous paper (Cai et et, 2014a), we showed the use of integrase-defective lentiviral vectors (IDLVs) as carriers of complete gene repair kits consisting of zinc-finger nuclease (ZFN) proteins and repair sequences, allowing gene correction by homologous recombination (HR). Here, we follow this strategy to engineer ZEN-loaded IDLVs that insert transgenes by a homology-driven mechanism into safe loci. This insertion mechanism is driven by time-restricted exposure of treated cells to ZFNs. We show targeted gene integration in human stem cells, including CD34+ hematopoietic progenitors and induced pluripotent stem cells (iPSCs). Notably, targeted insertions are identified in 89% of transduced iPSCs. Our findings demonstrate the applicability of nuclease-loaded 'all-in-one' IDLVs for site-directed gene insertion in stem cell based gene therapies.

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DO - 10.7554/eLife.12213

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JO - eLife

JF - eLife

M1 - e12213

ER -

Targeted, homology-driven gene insertion in stem cells by ZFN-loaded 'all-in-one' lentiviral vectors

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