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Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV

Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-...

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Autores principales: Naeimi Kararoudi, Meisam, Likhite, Shibi, Elmas, Ezgi, Yamamoto, Kenta, Schwartz, Maura, Sorathia, Kinnari, de Souza Fernandes Pereira, Marcelo, Sezgin, Yasemin, Devine, Raymond D., Lyberger, Justin M., Behbehani, Gregory K., Chakravarti, Nitin, Moriarity, Branden S., Meyer, Kathrin, Lee, Dean A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9243630/
https://www.ncbi.nlm.nih.gov/pubmed/35784645
http://dx.doi.org/10.1016/j.crmeth.2022.100236
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author Naeimi Kararoudi, Meisam
Likhite, Shibi
Elmas, Ezgi
Yamamoto, Kenta
Schwartz, Maura
Sorathia, Kinnari
de Souza Fernandes Pereira, Marcelo
Sezgin, Yasemin
Devine, Raymond D.
Lyberger, Justin M.
Behbehani, Gregory K.
Chakravarti, Nitin
Moriarity, Branden S.
Meyer, Kathrin
Lee, Dean A.
author_facet Naeimi Kararoudi, Meisam
Likhite, Shibi
Elmas, Ezgi
Yamamoto, Kenta
Schwartz, Maura
Sorathia, Kinnari
de Souza Fernandes Pereira, Marcelo
Sezgin, Yasemin
Devine, Raymond D.
Lyberger, Justin M.
Behbehani, Gregory K.
Chakravarti, Nitin
Moriarity, Branden S.
Meyer, Kathrin
Lee, Dean A.
author_sort Naeimi Kararoudi, Meisam
collection PubMed
description Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets.
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spelling pubmed-92436302022-07-01 Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV Naeimi Kararoudi, Meisam Likhite, Shibi Elmas, Ezgi Yamamoto, Kenta Schwartz, Maura Sorathia, Kinnari de Souza Fernandes Pereira, Marcelo Sezgin, Yasemin Devine, Raymond D. Lyberger, Justin M. Behbehani, Gregory K. Chakravarti, Nitin Moriarity, Branden S. Meyer, Kathrin Lee, Dean A. Cell Rep Methods Article Human primary natural killer (NK) cells are being widely advanced for cancer immunotherapy. However, methods for gene editing of these cells have suffered low transduction rates, high cell death, and loss of transgene expression after expansion. Here, we developed a highly efficient method for site-specific gene insertion in NK cells using CRISPR (Cas9/RNP) and AAVs. We compared AAV vectors designed to mediate gene insertion by different DNA repair mechanisms, homology arm lengths, and virus concentrations. We then validated the method for site-directed gene insertion of CD33-specific CARs into primary human NK cells. CAR transduction was efficient, its expression remained stable after expansion, and it improved efficacy against AML targets. Elsevier 2022-06-13 /pmc/articles/PMC9243630/ /pubmed/35784645 http://dx.doi.org/10.1016/j.crmeth.2022.100236 Text en © 2022 The Author(s) https://creativecommons.org/licenses/by-nc-nd/4.0/This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
spellingShingle Article
Naeimi Kararoudi, Meisam
Likhite, Shibi
Elmas, Ezgi
Yamamoto, Kenta
Schwartz, Maura
Sorathia, Kinnari
de Souza Fernandes Pereira, Marcelo
Sezgin, Yasemin
Devine, Raymond D.
Lyberger, Justin M.
Behbehani, Gregory K.
Chakravarti, Nitin
Moriarity, Branden S.
Meyer, Kathrin
Lee, Dean A.
Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title_full Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title_fullStr Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title_full_unstemmed Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title_short Optimization and validation of CAR transduction into human primary NK cells using CRISPR and AAV
title_sort optimization and validation of car transduction into human primary nk cells using crispr and aav
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9243630/
https://www.ncbi.nlm.nih.gov/pubmed/35784645
http://dx.doi.org/10.1016/j.crmeth.2022.100236
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