VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations
Background: In the context of ex vivo gene therapy or chimeric antigen receptor T cell (CAR-T) cell therapy, vector copy number (VCN) analysis in transduced cells by lentiviral vectors enables the assessment of risk and therapeutic efficiency in patients. Objectives: Here, we describe a ddPCR-b...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Mary Ann Liebert
2022-01-01
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| Series: | Re:GEN Open |
| Subjects: | |
| Online Access: | https://www.liebertpub.com/doi/full/10.1089/REGEN.2021.0034 |
| _version_ | 1797346070075801600 |
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| author | Aude Parcelier Julien Buisset Emilie Neveu Bastien Kauffmann Sandrine Fraboulet Peggy Sanatine Samia Martin C?drick Rousseaux Nadia Avenier Sabine Charrier |
| author_facet | Aude Parcelier Julien Buisset Emilie Neveu Bastien Kauffmann Sandrine Fraboulet Peggy Sanatine Samia Martin C?drick Rousseaux Nadia Avenier Sabine Charrier |
| author_sort | Aude Parcelier |
| collection | DOAJ |
| description |
Background: In the context of ex vivo gene therapy or chimeric antigen receptor T cell (CAR-T) cell therapy, vector copy number (VCN) analysis in transduced cells by lentiviral vectors enables the assessment of risk and therapeutic efficiency in patients.
Objectives:
Here, we describe a ddPCR-based method that can replace easily the TaqMan qPCR assay for VCN analysis, by measuring the number of pro-viral DNA copies per host cell genome in blood samples.
Methods: VCN are determined by ddPCR, after gDNA extraction and enzymatic digestion from transduced T lymphocytes or Hematopoietic Stem Cells (HSC) and by direct lysis of colony-forming cells (CFC) derived from transduced CD34+ cells.
Results: Firstly, we have identified key elements of sample preparation and set-up to further improve the performance characteristics of ddPCR method, resulting in an accurate analysis of VCN without the need for multiple replicates or an external calibrator, as required in qPCR methods. Secondly, we found that genomic DNA (gDNA) quantification by fluorometry allows a better prediction of the genomic copy number detected in ddPCR than by spectrophotometry. Then, we provide a new protocol to analyze VCN in blood cells but also in CFC, using a NaOH cell lysis-based approach.
Conclusion: This multiplex ddPCR is able to analyze VCN more precisely than qPCR in all transduced hematopoietic cells, an assay useful for clinical applications. |
| first_indexed | 2024-03-08T11:26:28Z |
| format | Article |
| id | doaj.art-1891b6ea0fe24f2fbad1c22dc45dcd7f |
| institution | Directory Open Access Journal |
| issn | 2766-2705 |
| language | English |
| last_indexed | 2024-03-08T11:26:28Z |
| publishDate | 2022-01-01 |
| publisher | Mary Ann Liebert |
| record_format | Article |
| series | Re:GEN Open |
| spelling | doaj.art-1891b6ea0fe24f2fbad1c22dc45dcd7f2024-01-26T05:13:31ZengMary Ann LiebertRe:GEN Open2766-27052022-01-0121283610.1089/REGEN.2021.0034VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and LimitationsAude Parcelier0Julien Buisset1Emilie Neveu2Bastien Kauffmann3Sandrine Fraboulet4Peggy Sanatine5Samia Martin6C?drick Rousseaux7Nadia Avenier8Sabine Charrier9YposkesiYposkesiYposkesiYposkesiYposkesiG?n?thonG?n?thonYposkesiYposkesiYposkesi Background: In the context of ex vivo gene therapy or chimeric antigen receptor T cell (CAR-T) cell therapy, vector copy number (VCN) analysis in transduced cells by lentiviral vectors enables the assessment of risk and therapeutic efficiency in patients. Objectives: Here, we describe a ddPCR-based method that can replace easily the TaqMan qPCR assay for VCN analysis, by measuring the number of pro-viral DNA copies per host cell genome in blood samples. Methods: VCN are determined by ddPCR, after gDNA extraction and enzymatic digestion from transduced T lymphocytes or Hematopoietic Stem Cells (HSC) and by direct lysis of colony-forming cells (CFC) derived from transduced CD34+ cells. Results: Firstly, we have identified key elements of sample preparation and set-up to further improve the performance characteristics of ddPCR method, resulting in an accurate analysis of VCN without the need for multiple replicates or an external calibrator, as required in qPCR methods. Secondly, we found that genomic DNA (gDNA) quantification by fluorometry allows a better prediction of the genomic copy number detected in ddPCR than by spectrophotometry. Then, we provide a new protocol to analyze VCN in blood cells but also in CFC, using a NaOH cell lysis-based approach. Conclusion: This multiplex ddPCR is able to analyze VCN more precisely than qPCR in all transduced hematopoietic cells, an assay useful for clinical applications.https://www.liebertpub.com/doi/full/10.1089/REGEN.2021.0034lentiviral vector copy numberddPCRgene therapyhematopoietic stem cells |
| spellingShingle | Aude Parcelier Julien Buisset Emilie Neveu Bastien Kauffmann Sandrine Fraboulet Peggy Sanatine Samia Martin C?drick Rousseaux Nadia Avenier Sabine Charrier VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations Re:GEN Open lentiviral vector copy number ddPCR gene therapy hematopoietic stem cells |
| title | VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations |
| title_full | VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations |
| title_fullStr | VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations |
| title_full_unstemmed | VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations |
| title_short | VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations |
| title_sort | vcn analysis using droplet digital pcr method in hematopoietic stem cells and t lymphocytes after lentiviral transduction optimization and limitations |
| topic | lentiviral vector copy number ddPCR gene therapy hematopoietic stem cells |
| url | https://www.liebertpub.com/doi/full/10.1089/REGEN.2021.0034 |
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