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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...

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Autores principales: Kaštánková, Iva, Štach, Martin, Žižková, Hana, Ptáčková, Pavlína, Šmilauerová, Kristýna, Mucha, Martin, Šroller, Vojtěch, Otáhal, Pavel
Formato: Online Artículo Texto
Lenguaje:English
Publicado: American Society of Gene & Cell Therapy 2021
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.
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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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