Sensor macrophages derived from human induced pluripotent stem cells to assess pyrogenic contaminations in parenteral drugs.

Ensuring the safety of parenteral drugs before injection into patients is of utmost importance. New regulations around the globe and the need to refrain from using animals however, have highlighted the need for new cell sources to be used in next-generation bioassays to detect the entire spectrum of possible contaminating pyrogens. Given the current drawbacks of the Monocyte-Activation-Test (MAT) with respect to the use of primary peripheral blood mono-nuclear cells or the use of monocytic cell lines, we here demonstrate the manufacturing of sensor monocytes/macrophages from human induced pluripotent stem cells (iMonoMac), which are fully defined and superior to current cell products. Using a modern and scalable manufacturing platform, iMonoMac showed typical macrophage-like morphology and stained positive for several Toll like receptor (TLRs) such as TLR-2, TLR-5, TLR-4. Furthermore, iMonoMac derived from the same donor were sensitive to endotoxins, non-endotoxins, and process related pyrogens at a high dynamic range and across different cellular densities. Of note, iMonoMac showed increased sensitivity and reactivity to a broad range of pyrogens, demonstrated by the detection of interleukin-6 at low concentrations of LPS and MALP-2 which could not be reached using the current MAT cell sources. To further advance the system, iMonoMac or genetically engineered iMonoMac with NF-κB-luciferase reporter cassette could reveal a specific activation response while correlating to the classical detection method employing enzyme-linked immunosorbent assay to measure cytokine secretion. Thus, we present a valuable cellular tool to assess parenteral drugs safety, facilitating the future acceptance and design of regulatory-approved bioassays.

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

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 (mHSPCs) 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 mHSPCs 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 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.