Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products
BACKGROUND: Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies.
METHODS: A digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells.
RESULTS: The ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results.
CONCLUSIONS: ddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2020 |
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| Erschienen: |
2020 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:18 |
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| Enthalten in: |
Journal of translational medicine - 18(2020), 1 vom: 08. Mai, Seite 191 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Lu, Alex [VerfasserIn] |
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| Links: |
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| Anmerkungen: |
Date Completed 14.05.2021 Date Revised 14.05.2021 published: Electronic Citation Status MEDLINE |
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| doi: |
10.1186/s12967-020-02358-0 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM309681456 |
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| 100 | 1 | |a Lu, Alex |e verfasserin |4 aut | |
| 245 | 1 | 0 | |a Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
| 264 | 1 | |c 2020 | |
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| 500 | |a Date Revised 14.05.2021 | ||
| 500 | |a published: Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a BACKGROUND: Genetically engineered T cells have become an important therapy for B-cell malignancies. Measuring the efficiency of vector integration into the T cell genome is important for assessing the potency and safety of these cancer immunotherapies | ||
| 520 | |a METHODS: A digital droplet polymerase chain reaction (ddPCR) assay was developed and evaluated for assessing the average number of lenti- and retroviral vectors integrated into Chimeric Antigen Receptor (CAR) and T Cell Receptor (TCR)-engineered T cells | ||
| 520 | |a RESULTS: The ddPCR assay consistently measured the concentration of an empty vector in solution and the average number of CAR and TCR vectors integrated into T cell populations. There was a linear relationship between the average vector copy number per cell measured by ddPCR and the proportion of cells transduced as measured by flow cytometry. Similar vector copy number measurements were obtained by different staff using the ddPCR assay, highlighting the assays reproducibility among technicians. Analysis of fresh and cryopreserved CAR T and TCR engineered T cells yielded similar results | ||
| 520 | |a CONCLUSIONS: ddPCR is a robust tool for accurate quantitation of average vector copy number in CAR and TCR engineered T cells. The assay is also applicable to other types of genetically engineered cells including Natural Killer cells and hematopoietic stem cells | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, N.I.H., Intramural | |
| 650 | 4 | |a Cellular cancer immunotherapy | |
| 650 | 4 | |a Chimeric antigen receptor (CAR) T cells | |
| 650 | 4 | |a Droplet digital PCR | |
| 650 | 4 | |a Gene therapy | |
| 650 | 4 | |a Genetically engineered T cells | |
| 650 | 4 | |a T Cell Receptor (TCR)-engineered T cells | |
| 650 | 4 | |a Vector copy number | |
| 650 | 7 | |a Receptors, Chimeric Antigen |2 NLM | |
| 700 | 1 | |a Liu, Hui |e verfasserin |4 aut | |
| 700 | 1 | |a Shi, Rongye |e verfasserin |4 aut | |
| 700 | 1 | |a Cai, Yihua |e verfasserin |4 aut | |
| 700 | 1 | |a Ma, Jinxia |e verfasserin |4 aut | |
| 700 | 1 | |a Shao, Lipei |e verfasserin |4 aut | |
| 700 | 1 | |a Rong, Victor |e verfasserin |4 aut | |
| 700 | 1 | |a Gkitsas, Nikolaos |e verfasserin |4 aut | |
| 700 | 1 | |a Lei, Hong |e verfasserin |4 aut | |
| 700 | 1 | |a Highfill, Steven L |e verfasserin |4 aut | |
| 700 | 1 | |a Panch, Sandhya |e verfasserin |4 aut | |
| 700 | 1 | |a Stroncek, David F |e verfasserin |4 aut | |
| 700 | 1 | |a Jin, Ping |e verfasserin |4 aut | |
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