Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products
Abstract 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 c...
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doaj-2235df50030c405a9bd527395ca064bf2020-11-25T02:04:33ZengBMCJournal of Translational Medicine1479-58762020-05-011811710.1186/s12967-020-02358-0Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell productsAlex Lu0Hui Liu1Rongye Shi2Yihua Cai3Jinxia Ma4Lipei Shao5Victor Rong6Nikolaos Gkitsas7Hong Lei8Steven L. Highfill9Sandhya Panch10David F. Stroncek11Ping Jin12Center for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterCenter for Cellular Engineering, Department of Transfusion Medicine and Cellular Engineering, NIH Clinical CenterAbstract 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.http://link.springer.com/article/10.1186/s12967-020-02358-0Droplet digital PCRVector copy numberGenetically engineered T cellsGene therapyChimeric antigen receptor (CAR) T cellsT Cell Receptor (TCR)-engineered T cells |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Alex Lu Hui Liu Rongye Shi Yihua Cai Jinxia Ma Lipei Shao Victor Rong Nikolaos Gkitsas Hong Lei Steven L. Highfill Sandhya Panch David F. Stroncek Ping Jin |
spellingShingle |
Alex Lu Hui Liu Rongye Shi Yihua Cai Jinxia Ma Lipei Shao Victor Rong Nikolaos Gkitsas Hong Lei Steven L. Highfill Sandhya Panch David F. Stroncek Ping Jin Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products Journal of Translational Medicine Droplet digital PCR Vector copy number Genetically engineered T cells Gene therapy Chimeric antigen receptor (CAR) T cells T Cell Receptor (TCR)-engineered T cells |
author_facet |
Alex Lu Hui Liu Rongye Shi Yihua Cai Jinxia Ma Lipei Shao Victor Rong Nikolaos Gkitsas Hong Lei Steven L. Highfill Sandhya Panch David F. Stroncek Ping Jin |
author_sort |
Alex Lu |
title |
Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
title_short |
Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
title_full |
Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
title_fullStr |
Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
title_full_unstemmed |
Application of droplet digital PCR for the detection of vector copy number in clinical CAR/TCR T cell products |
title_sort |
application of droplet digital pcr for the detection of vector copy number in clinical car/tcr t cell products |
publisher |
BMC |
series |
Journal of Translational Medicine |
issn |
1479-5876 |
publishDate |
2020-05-01 |
description |
Abstract 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. |
topic |
Droplet digital PCR Vector copy number Genetically engineered T cells Gene therapy Chimeric antigen receptor (CAR) T cells T Cell Receptor (TCR)-engineered T cells |
url |
http://link.springer.com/article/10.1186/s12967-020-02358-0 |
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