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Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells
The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high cos...
Autores principales: | , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society of Gene & Cell Therapy
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482285/ https://www.ncbi.nlm.nih.gov/pubmed/34631931 http://dx.doi.org/10.1016/j.omtm.2021.08.006 |
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author | Kaštánková, Iva Štach, Martin Žižková, Hana Ptáčková, Pavlína Šmilauerová, Kristýna Mucha, Martin Šroller, Vojtěch Otáhal, Pavel |
author_facet | Kaštánková, Iva Štach, Martin Žižková, Hana Ptáčková, Pavlína Šmilauerová, Kristýna Mucha, Martin Šroller, Vojtěch Otáhal, Pavel |
author_sort | Kaštánková, Iva |
collection | PubMed |
description | The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3′ and 5′ arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 10(8) CAR19 T cells per one electroporation of 1 × 10(7) peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products. |
format | Online Article Text |
id | pubmed-8482285 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | American Society of Gene & Cell Therapy |
record_format | MEDLINE/PubMed |
spelling | pubmed-84822852021-10-08 Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells Kaštánková, Iva Štach, Martin Žižková, Hana Ptáčková, Pavlína Šmilauerová, Kristýna Mucha, Martin Šroller, Vojtěch Otáhal, Pavel Mol Ther Methods Clin Dev Original Article The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3′ and 5′ arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 10(8) CAR19 T cells per one electroporation of 1 × 10(7) peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products. American Society of Gene & Cell Therapy 2021-08-26 /pmc/articles/PMC8482285/ /pubmed/34631931 http://dx.doi.org/10.1016/j.omtm.2021.08.006 Text en © 2021 The Authors 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 | Original Article Kaštánková, Iva Štach, Martin Žižková, Hana Ptáčková, Pavlína Šmilauerová, Kristýna Mucha, Martin Šroller, Vojtěch Otáhal, Pavel Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title | Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title_full | Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title_fullStr | Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title_full_unstemmed | Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title_short | Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells |
title_sort | enzymatically produced piggybac transposon vectors for efficient non-viral manufacturing of cd19-specific car t cells |
topic | Original Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8482285/ https://www.ncbi.nlm.nih.gov/pubmed/34631931 http://dx.doi.org/10.1016/j.omtm.2021.08.006 |
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