Abstract

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual 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 Invitrogen’s flow 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 number 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 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.

Highlights

  • Stem cell-based gene therapy is a promising area of medicine that is rapidly expanding with regards to clinical trials and marketing authorization

  • Gene therapy for immunodeficiencies is commonly performed by inserting a normal copy of the defective gene into the patient’s CD34+ enriched HSPCs isolated from bone marrow (BM) or mobilized peripheral blood

  • Cells were transduced with the same number of viral particles per cell (VP/cell) and infectious genomes (MOI) (1000 VP/cell; MOI 3.6), a different transduction efficiency can be observed both by the percentage of transduced cells and the mean fluorescent intensity (MFI) with different values for Cord Blood (CB) (14.8%; 16.4 MFI), Bone Marrow (BM) cells (16.1%; 9.03 MFI) and Mobilized Peripheral Blood (mPB) cells (15%; 15.01 MFI), indicating.differences in cell permissiveness of human HSPCs depending on the source

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Summary

Introduction

Stem cell-based gene therapy is a promising area of medicine that is rapidly expanding with regards to clinical trials and marketing authorization. The first ex vivo 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) [1,2,3,4] This paved the way for the clinical development of HSC-based gene therapy to treat other immunodeficiencies, including the SCIDs X-linked [5,6,7,8,9], Artemis [10,11,12,13], RAG1 [14], as well as X-linked chronic granulomatous disease (CGD) [15, 16] and Wiskott–Aldrich Syndrome (WAS) [17,18,19,20,21]. VCN is an important parameter to control because multiple vector copies per cell can result in genotoxicity

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