An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products
Background: Genetically engineered T-cell therapy holds immense promise in cancer immunotherapy. These T-cell products are typically engineered by vectors that permanently integrate into the T-cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes...
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Elsevier
2025-03-01
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| Series: | Immuno-Oncology and Technology |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2590018824003289 |
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| author | J. Ma S. Meyer J. Olweus C. Jin D. Yu |
| author_facet | J. Ma S. Meyer J. Olweus C. Jin D. Yu |
| author_sort | J. Ma |
| collection | DOAJ |
| description | Background: Genetically engineered T-cell therapy holds immense promise in cancer immunotherapy. These T-cell products are typically engineered by vectors that permanently integrate into the T-cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes imperative to assess the integrated vector copy number (VCN) as a critical safety parameter for gene-engineered cell products. Materials and methods: In this study, we developed a robust assay for assessing the VCN of chimeric antigen receptor-T cell and T-cell receptor T-cell products, based on the droplet digital polymerase chain reaction (ddPCR) method. To provide accurate representation of the VCN in gene-engineered cells, we implemented a calculation that factors in the putative transduction efficiency based on Poisson distribution statistics. The adjusted VCN value (VCNadj) was also compared with VCN value from sorted transgene-positive cell populations, to validate its accuracy. Results: This assay consistently and accurately determines the average VCN for cell products. By comparing the VCN in sorted transgene-positive cell populations, we validated the refinement calculation provides a closer approximation to the actual VCN within transduced cells, offering a more realistic representation of the VCN for engineered cell products. Conclusion: In summary, we present a reliable and robust ddPCR-based assay for quantification of VCN in gene-engineered cell products. |
| format | Article |
| id | doaj-art-46cab4a4f08f4046bbb17c7e13fc56aa |
| institution | Kabale University |
| issn | 2590-0188 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
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| series | Immuno-Oncology and Technology |
| spelling | doaj-art-46cab4a4f08f4046bbb17c7e13fc56aa2025-08-20T03:42:26ZengElsevierImmuno-Oncology and Technology2590-01882025-03-012510103110.1016/j.iotech.2024.101031An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell productsJ. Ma0S. Meyer1J. Olweus2C. Jin3D. Yu4Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; Elicera Therapeutics AB, Gothenburg, SwedenDepartment of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway; Precision Immunotherapy Alliance, University of Oslo, Oslo, NorwayDepartment of Cancer Immunology, Institute for Cancer Research, Oslo University Hospital Radiumhospitalet, Oslo, Norway; Precision Immunotherapy Alliance, University of Oslo, Oslo, NorwayDepartment of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; Elicera Therapeutics AB, Gothenburg, Sweden; Correspondence to: Dr Chuan Jin, Rudbecklaboratoriet, Dag Hammarskjölds Väg 20, 751 85 Uppsala, Sweden. Tel: +46(0)18-4710000Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden; Elicera Therapeutics AB, Gothenburg, Sweden; Associate Prof Di Yu, Rudbecklaboratoriet, Dag Hammarskjölds Väg 20, 751 85 Uppsala, Sweden. Tel: +46(0)18-4710000Background: Genetically engineered T-cell therapy holds immense promise in cancer immunotherapy. These T-cell products are typically engineered by vectors that permanently integrate into the T-cell genome, thus raising concerns about potential risks of insertional mutagenesis. Therefore, it becomes imperative to assess the integrated vector copy number (VCN) as a critical safety parameter for gene-engineered cell products. Materials and methods: In this study, we developed a robust assay for assessing the VCN of chimeric antigen receptor-T cell and T-cell receptor T-cell products, based on the droplet digital polymerase chain reaction (ddPCR) method. To provide accurate representation of the VCN in gene-engineered cells, we implemented a calculation that factors in the putative transduction efficiency based on Poisson distribution statistics. The adjusted VCN value (VCNadj) was also compared with VCN value from sorted transgene-positive cell populations, to validate its accuracy. Results: This assay consistently and accurately determines the average VCN for cell products. By comparing the VCN in sorted transgene-positive cell populations, we validated the refinement calculation provides a closer approximation to the actual VCN within transduced cells, offering a more realistic representation of the VCN for engineered cell products. Conclusion: In summary, we present a reliable and robust ddPCR-based assay for quantification of VCN in gene-engineered cell products.http://www.sciencedirect.com/science/article/pii/S2590018824003289droplet digital PCR (ddPCR)vector copy number (VCN)genetically engineered T cells |
| spellingShingle | J. Ma S. Meyer J. Olweus C. Jin D. Yu An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products Immuno-Oncology and Technology droplet digital PCR (ddPCR) vector copy number (VCN) genetically engineered T cells |
| title | An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products |
| title_full | An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products |
| title_fullStr | An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products |
| title_full_unstemmed | An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products |
| title_short | An adjusted droplet digital PCR assay for quantification of vector copy number in CAR-T cell and TCR-T cell products |
| title_sort | adjusted droplet digital pcr assay for quantification of vector copy number in car t cell and tcr t cell products |
| topic | droplet digital PCR (ddPCR) vector copy number (VCN) genetically engineered T cells |
| url | http://www.sciencedirect.com/science/article/pii/S2590018824003289 |
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