Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases

Mark J. Osborn; Beau R. Webber; Friederike Knipping; Cara Lin Lonetree; Nicole Tennis; Anthony P. DeFeo; Amber N. McElroy; Colby G. Starker; Catherine Lee; Sarah Merkel; Troy C. Lund; Karen S. Kelly-Spratt; Michael C. Jensen; Daniel F. Voytas; Christof Von Kalle; Manfred Schmidt; Richard Gabriel; Keli L. Hippen; Jeffrey S. Miller; Andrew M. Scharenberg; Jakub Tolar; Bruce R. Blazar

doi:10.1038/mt.2015.197

Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases

Mark J. Osborn, Beau R. Webber, Friederike Knipping, Cara Lin Lonetree, Nicole Tennis, Anthony P. DeFeo, Amber N. McElroy, Colby G. Starker, Catherine Lee, Sarah Merkel, Troy C. Lund, Karen S. Kelly-Spratt, Michael C. Jensen, Daniel F. Voytas, Christof Von Kalle, Manfred Schmidt, Richard Gabriel, Keli L. Hippen, Jeffrey S. Miller, Andrew M. ScharenbergJakub Tolar, Bruce R. Blazar

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

149 Scopus citations

Abstract

Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.

Original language	English (US)
Pages (from-to)	570-581
Number of pages	12
Journal	Molecular Therapy
Volume	24
Issue number	3
DOIs	https://doi.org/10.1038/mt.2015.197
State	Published - Mar 1 2016

Bibliographical note

Funding Information:
The authors are grateful to Kelsey Vigoren for editorial help with this article. The authors are also grateful to Jordan Jarjour and Alexander Astrakhan for helpful comments and edits. The authors appreciate funding support from The Children’s Cancer Research Fund, the Lindahl Family & the Corrigan Family, and the Masonic Cancer Center Cancer Experimental Therapeutics Initiative. B.R.W. is supported by NIH T32-HL007062. J.T. is supported in part by R01 AR063070 and P01 CA065493. M.J.O. is supported by 8UL1TR000114-02. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Funding Information:
The authors are grateful to Kelsey Vigoren for editorial help with this article. The authors are also grateful to Jordan Jarjour and Alexander Astrakhan for helpful comments and edits. The authors appreciate funding support from The Children''s Cancer Research Fund, the Lindahl Family & the Corrigan Family, and the Masonic Cancer Center Cancer Experimental Therapeutics Initiative. B.R.W. is supported by NIH T32-HL007062. J.T. is supported in part by R01 AR063070 and P01 CA065493. M.J.O. is supported by 8UL1TR000114-02. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2016 The American Society of Gene & Cell Therapy.

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Osborn, M. J., Webber, B. R., Knipping, F., Lonetree, C. L., Tennis, N., DeFeo, A. P., McElroy, A. N., Starker, C. G., Lee, C., Merkel, S., Lund, T. C., Kelly-Spratt, K. S., Jensen, M. C., Voytas, D. F., Von Kalle, C., Schmidt, M., Gabriel, R., Hippen, K. L., Miller, J. S., ... Blazar, B. R. (2016). Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases. Molecular Therapy, 24(3), 570-581. https://doi.org/10.1038/mt.2015.197

Osborn, MJ , Webber, BR, Knipping, F, Lonetree, CL, Tennis, N, DeFeo, AP , McElroy, AN , Starker, CG, Lee, C, Merkel, S, Lund, TC, Kelly-Spratt, KS, Jensen, MC, Voytas, DF, Von Kalle, C, Schmidt, M, Gabriel, R, Hippen, KL , Miller, JS, Scharenberg, AM, Tolar, J & Blazar, BR 2016, 'Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases', Molecular Therapy, vol. 24, no. 3, pp. 570-581. https://doi.org/10.1038/mt.2015.197

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abstract = "Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.",

author = "Osborn, {Mark J.} and Webber, {Beau R.} and Friederike Knipping and Lonetree, {Cara Lin} and Nicole Tennis and DeFeo, {Anthony P.} and McElroy, {Amber N.} and Starker, {Colby G.} and Catherine Lee and Sarah Merkel and Lund, {Troy C.} and Kelly-Spratt, {Karen S.} and Jensen, {Michael C.} and Voytas, {Daniel F.} and {Von Kalle}, Christof and Manfred Schmidt and Richard Gabriel and Hippen, {Keli L.} and Miller, {Jeffrey S.} and Scharenberg, {Andrew M.} and Jakub Tolar and Blazar, {Bruce R.}",

note = "Funding Information: The authors are grateful to Kelsey Vigoren for editorial help with this article. The authors are also grateful to Jordan Jarjour and Alexander Astrakhan for helpful comments and edits. The authors appreciate funding support from The Children{\textquoteright}s Cancer Research Fund, the Lindahl Family & the Corrigan Family, and the Masonic Cancer Center Cancer Experimental Therapeutics Initiative. B.R.W. is supported by NIH T32-HL007062. J.T. is supported in part by R01 AR063070 and P01 CA065493. M.J.O. is supported by 8UL1TR000114-02. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Funding Information: The authors are grateful to Kelsey Vigoren for editorial help with this article. The authors are also grateful to Jordan Jarjour and Alexander Astrakhan for helpful comments and edits. The authors appreciate funding support from The Children''s Cancer Research Fund, the Lindahl Family & the Corrigan Family, and the Masonic Cancer Center Cancer Experimental Therapeutics Initiative. B.R.W. is supported by NIH T32-HL007062. J.T. is supported in part by R01 AR063070 and P01 CA065493. M.J.O. is supported by 8UL1TR000114-02. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health Award Number UL1TR000114 (M.J.O.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. Publisher Copyright: {\textcopyright} 2016 The American Society of Gene & Cell Therapy.",

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doi = "10.1038/mt.2015.197",

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AU - Osborn, Mark J.

AU - Webber, Beau R.

AU - Knipping, Friederike

AU - Lonetree, Cara Lin

AU - Tennis, Nicole

AU - DeFeo, Anthony P.

AU - McElroy, Amber N.

AU - Starker, Colby G.

AU - Lee, Catherine

AU - Merkel, Sarah

AU - Lund, Troy C.

AU - Kelly-Spratt, Karen S.

AU - Jensen, Michael C.

AU - Voytas, Daniel F.

AU - Von Kalle, Christof

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AU - Gabriel, Richard

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AU - Scharenberg, Andrew M.

AU - Tolar, Jakub

AU - Blazar, Bruce R.

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N2 - Present adoptive immunotherapy strategies are based on the re-targeting of autologous T-cells to recognize tumor antigens. As T-cell properties may vary significantly between patients, this approach can result in significant variability in cell potency that may affect therapeutic outcome. More consistent results could be achieved by generating allogeneic cells from healthy donors. An impediment to such an approach is the endogenous T-cell receptors present on T-cells, which have the potential to direct dangerous off-tumor antihost reactivity. To address these limitations, we assessed the ability of three different TCR-α-targeted nucleases to disrupt T-cell receptor expression in primary human T-cells. We optimized the conditions for the delivery of each reagent and assessed off-target cleavage. The megaTAL and CRISPR/Cas9 reagents exhibited the highest disruption efficiency combined with low levels of toxicity and off-target cleavage, and we used them for a translatable manufacturing process to produce safe cellular substrates for next-generation immunotherapies.

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Evaluation of TCR gene editing achieved by TALENs, CRISPR/Cas9, and megaTAL nucleases

Abstract

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