Development of an in vitro genotoxicity assay to detect retroviral vector-induced lymphoid insertional mutants [bulk]

Safety assessment in retroviral vector-mediated gene therapy remains challenging. In clinical trials for different blood and immune disorders, insertional mutagenesis led to myeloid and lymphoid leukemia. We previously developed the In Vitro Immortalization Assay (IVIM) and Surrogate Assay for Genotoxicity Assessment (SAGA) for pre-clinical genotoxicity prediction of integrating vectors. Murine hematopoietic stem and progenitor cells (mHSPC) transduced with mutagenic vectors acquire a proliferation advantage under limiting dilution (IVIM) and activate stem cell- and cancer-related transcriptional programs (SAGA). However, both assays present an intrinsic myeloid bias due to culture conditions. To detect lymphoid mutants, we differentiated mHSPC to mature T cells and analyzed their phenotype, insertion site pattern, and gene expression changes after transduction with retroviral vectors. Mutagenic vectors induced a block in differentiation at an early progenitor stage (double-negative 2) compared to fully differentiated untransduced mock cultures. Arrested samples harbored high-risk insertions close to Lmo2, frequently observed in clinical trials with severe adverse events. Lymphoid insertional mutants displayed a unique gene expression signature identified by the machine learning algorithm of SAGA. The gene expression-based highly sensitive molecular readout will broaden our understanding of vector-induced oncogenicity and help in pre-clinical prediction of retroviral genotoxicity. Murine lineage negative bone marrow cells were isolated from C57BL/6J mice, transduced with different integrating retroviral vectors and cultured in lymphoid cytokine conditions for up to 54 days. The current study comprises of eight independent experiments. In each experiment, all the different vector types and mock samples (untransduced controls) were tested in triplicates. At different time points, cells were harvested from the bulk cultures, RNA was isolated and used for microarray analysis. The RNA was hybridized to Agilent ID 084956, which is a custom refined version of the Agilent Whole Mouse Genome Oligo Microarray 4x44k v2 (Design ID 026655, Agilent Techonologies), developed by the Research Core Unit Genomics (RCUG) of Hannover Medical School (GEO accession GPL27545). All non-control probes of design ID 026655 have been printed four times within a region comprising a total of 181560 Features (170 columns x 1068 rows). Four of such regions were placed within one 1M region giving rise to four microarray fields per slide to be hybridized individually (Customer Specified Feature Layout). Data extraction was performed with the ‘FeatureExtraction Software V10.7.3.1’ using the extraction protocol file ‘GE1_107_Sep09.xml’.