A highly efficient transgene knock-in technology in clinically relevant cell types

Alexander G. Allen; Samia Q. Khan; Carrie M. Margulies; Ramya Viswanathan; Swarali Lele; Laura Blaha; Sean N. Scott; Kaitlyn M. Izzo; Alexandra Gerew; Rithu Pattali; Nadire R. Cochran; Carl S. Holland; Amy H. Zhao; Stephen E. Sherman; Michael C. Jaskolka; Meng Wu; Aaron C. Wilson; Xiaoqi Sun; Dawn M. Ciulla; Deric Zhang; Jacqueline D. Nelson; Peisheng Zhang; Patrizia Mazzucato; Yan Huang; Georgia Giannoukos; Eugenio Marco; Michael Nehil; John A. Follit; Kai Hsin Chang; Mark S. Shearman; Christopher J. Wilson; John A. Zuris

doi:10.1038/s41587-023-01779-8

A highly efficient transgene knock-in technology in clinically relevant cell types

Alexander G. Allen, Samia Q. Khan, Carrie M. Margulies, Ramya Viswanathan, Swarali Lele, Laura Blaha, Sean N. Scott, Kaitlyn M. Izzo, Alexandra Gerew, Rithu Pattali, Nadire R. Cochran, Carl S. Holland, Amy H. Zhao, Stephen E. Sherman, Michael C. Jaskolka, Meng Wu, Aaron C. Wilson, Xiaoqi Sun, Dawn M. Ciulla, Deric ZhangJacqueline D. Nelson, Peisheng Zhang, Patrizia Mazzucato, Yan Huang, Georgia Giannoukos, Eugenio Marco, Michael Nehil, John A. Follit, Kai Hsin Chang, Mark S. Shearman, Christopher J. Wilson, John A. Zuris

Research output: Contribution to journal › Article › peer-review

26 Scopus citations

Abstract

Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

Original language	English
Pages (from-to)	458-469
Number of pages	12
Journal	Nature Biotechnology
Volume	42
Issue number	3
DOIs	https://doi.org/10.1038/s41587-023-01779-8
State	Published - Mar 2024
Externally published	Yes

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Allen, A. G., Khan, S. Q., Margulies, C. M., Viswanathan, R., Lele, S., Blaha, L., Scott, S. N., Izzo, K. M., Gerew, A., Pattali, R., Cochran, N. R., Holland, C. S., Zhao, A. H., Sherman, S. E., Jaskolka, M. C., Wu, M., Wilson, A. C., Sun, X., Ciulla, D. M., ... Zuris, J. A. (2024). A highly efficient transgene knock-in technology in clinically relevant cell types. Nature Biotechnology, 42(3), 458-469. https://doi.org/10.1038/s41587-023-01779-8

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title = "A highly efficient transgene knock-in technology in clinically relevant cell types",

abstract = "Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.",

author = "Allen, {Alexander G.} and Khan, {Samia Q.} and Margulies, {Carrie M.} and Ramya Viswanathan and Swarali Lele and Laura Blaha and Scott, {Sean N.} and Izzo, {Kaitlyn M.} and Alexandra Gerew and Rithu Pattali and Cochran, {Nadire R.} and Holland, {Carl S.} and Zhao, {Amy H.} and Sherman, {Stephen E.} and Jaskolka, {Michael C.} and Meng Wu and Wilson, {Aaron C.} and Xiaoqi Sun and Ciulla, {Dawn M.} and Deric Zhang and Nelson, {Jacqueline D.} and Peisheng Zhang and Patrizia Mazzucato and Yan Huang and Georgia Giannoukos and Eugenio Marco and Michael Nehil and Follit, {John A.} and Chang, {Kai Hsin} and Shearman, {Mark S.} and Wilson, {Christopher J.} and Zuris, {John A.}",

note = "Publisher Copyright: {\textcopyright} The Author(s), under exclusive licence to Springer Nature America, Inc. 2023.",

year = "2024",

month = mar,

doi = "10.1038/s41587-023-01779-8",

language = "English",

volume = "42",

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Allen, AG, Khan, SQ, Margulies, CM, Viswanathan, R, Lele, S, Blaha, L, Scott, SN, Izzo, KM, Gerew, A, Pattali, R, Cochran, NR, Holland, CS, Zhao, AH, Sherman, SE, Jaskolka, MC, Wu, M, Wilson, AC, Sun, X, Ciulla, DM, Zhang, D, Nelson, JD, Zhang, P, Mazzucato, P, Huang, Y, Giannoukos, G, Marco, E, Nehil, M, Follit, JA, Chang, KH, Shearman, MS, Wilson, CJ & Zuris, JA 2024, 'A highly efficient transgene knock-in technology in clinically relevant cell types', Nature Biotechnology, vol. 42, no. 3, pp. 458-469. https://doi.org/10.1038/s41587-023-01779-8

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AU - Allen, Alexander G.

AU - Khan, Samia Q.

AU - Margulies, Carrie M.

AU - Viswanathan, Ramya

AU - Lele, Swarali

AU - Blaha, Laura

AU - Scott, Sean N.

AU - Izzo, Kaitlyn M.

AU - Gerew, Alexandra

AU - Pattali, Rithu

AU - Cochran, Nadire R.

AU - Holland, Carl S.

AU - Zhao, Amy H.

AU - Sherman, Stephen E.

AU - Jaskolka, Michael C.

AU - Wu, Meng

AU - Wilson, Aaron C.

AU - Sun, Xiaoqi

AU - Ciulla, Dawn M.

AU - Zhang, Deric

AU - Nelson, Jacqueline D.

AU - Zhang, Peisheng

AU - Mazzucato, Patrizia

AU - Huang, Yan

AU - Giannoukos, Georgia

AU - Marco, Eugenio

AU - Nehil, Michael

AU - Follit, John A.

AU - Chang, Kai Hsin

AU - Shearman, Mark S.

AU - Wilson, Christopher J.

AU - Zuris, John A.

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AB - Inefficient knock-in of transgene cargos limits the potential of cell-based medicines. In this study, we used a CRISPR nuclease that targets a site within an exon of an essential gene and designed a cargo template so that correct knock-in would retain essential gene function while also integrating the transgene(s) of interest. Cells with non-productive insertions and deletions would undergo negative selection. This technology, called SLEEK (SeLection by Essential-gene Exon Knock-in), achieved knock-in efficiencies of more than 90% in clinically relevant cell types without impacting long-term viability or expansion. SLEEK knock-in rates in T cells are more efficient than state-of-the-art TRAC knock-in with AAV6 and surpass more than 90% efficiency even with non-viral DNA cargos. As a clinical application, natural killer cells generated from induced pluripotent stem cells containing SLEEK knock-in of CD16 and mbIL-15 show substantially improved tumor killing and persistence in vivo.

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A highly efficient transgene knock-in technology in clinically relevant cell types

Abstract

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