Measuring the distribution of vector copies in corrected cells at a single-cell level is usually important to assess that this actual VCN is in the therapeutic range of sufficient integration without the risk of genotoxicity. VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogens circulation cytometry-based PrimeFlow? RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow?-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the quantity of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available 5′-GTP trisodium salt hydrate or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells. gene therapy product using autologous HSC for the treatment of Adenosine Deaminase (ADA) Severe Combined Immunodeficiency (SCID) was Strimvelis (GlaxoSmithKline), and was approved in 2016 by the European Medicines Agency (EMA) (1C4). This paved the way for the clinical development of HSC-based gene therapy to treat other immunodeficiencies, including the SCIDs X-linked (5C9), Artemis (10C13), RAG1 (14), as well as X-linked chronic granulomatous disease (CGD) (15, 16) and WiskottCAldrich Syndrome (WAS) (17C21). For the treatment of main immunodeficiencies, hematopoietic stem and progenitor 5′-GTP trisodium salt hydrate cells (HSPCs) are corrected (examined by Staal et?al. (22). The patients HSPCs are isolated, altered with the therapeutic transgene by viral transduction and the corrected cells, the gene therapy product, are transplanted back into the patient. Genome-integrating vectors like self-inactivating (SIN) gamma-retroviral (-RV) and lentiviral vectors (LV) have been used safely over the past two decades to achieve a long-lasting therapeutic effect of the transgene (23). One of the main release criteria for the treatment with a gene therapy product is LENG8 antibody to reach sufficient transgene expression, measured by the number of integrated transgene copies per target cell known as the vector copy number 5′-GTP trisodium salt hydrate (VCN). The therapeutic potency of the transgene correlates positively with the proportion of transduced cells, therefore, a threshold is set for the minimal transduction efficiency required to assurance the correction of enough cells with sufficient transgene expression for a successful and safe therapeutic outcome. However, VCN is an important parameter to control because multiple vector copies per cell can result in 5′-GTP trisodium salt hydrate genotoxicity. The golden standard technique to reliably measure VCN has been quantitative polymerase chain reaction (qPCR) of a LV sequence relative to a housekeeping gene to determine the number of inserted vectors (24). This strategy determines the average VCN per cell in the bulk population, while only a proportion of cells carry the 5′-GTP trisodium salt hydrate therapeutic vector. Therefore, the presence of non-transduced cells in the bulk populace invariably underestimates the VCN of the therapeutic cells. Measuring the distribution of vector copies in corrected cells at a single-cell level is usually important to assess that this actual VCN is in the therapeutic range of sufficient integration without the risk of genotoxicity. Attempts to refrain from the bulk populace average have been accomplished by measuring VCN in individual colony-forming cell models (CFC). Transduction efficiency determination in CFC has developed from a green fluorescent protein (GFP) detection method (25) to more reliable and simplified qPCR assays (26, 27) that have been further validated with clinically relevant experimental data (28). Although this strategy is a step forward towards a better understanding of the cellular heterogeneity of the therapeutic product, proper single cell information is still missing. Thus, determining transgene expression with a multiparametric technology such as circulation cytometry represents a quick single-cell alternative to CFC assays and a stylish alternative to bulk methods. Here, we expose a method based on the PrimeFlow RNA Assay? (Thermo Fisher Scientific) (29, 30) generally referred to as Branched DNA method, as a potential new tool to.