VCN Analysis Using Droplet Digital PCR Method in Hematopoietic Stem Cells and T Lymphocytes after Lentiviral Transduction: Optimization and Limitations

Abstract

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.

Keywords

• lentiviral vector copy number
• ddPCR
• gene therapy
• hematopoietic stem cells