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

Biomanufacturing in gene and cell therapy

“… 9 address critical safety concerns associated with retroviral vectors, known for their risk of insertional mutagenesis which has led to leukemia in some gene therapy recipients. 9 , 10 They developed an in vitro genotoxicity assay using murine hematopoietic stem and progenitor cells to detect harmful mutations induced by retroviral vectors, particularly those affecting lymphoid cells. This assay aims to improve safety by identifying potentially oncogenic changes early in the vector development process.…”

Advancements in Hematopoietic Stem Cell Gene Therapy: A Journey of Progress for Viral Transduction

“…Additionally, in vitro assays such as the In Vitro Immortalization assay (IVIM) and the Surrogate Assay for Genotoxicity Assessment (SAGA) have been developed in an effort to predict or quantify the pre-clinical genotoxicity of integrating vectors. Notably, IVIM quantifies the mutagenic potential of retroviruses based on the acquisition of a proliferation advantage under limiting dilution conditions of murine hematopoietic stem and progenitor cells transduced with mutagenic vectors [ 54 ]. Although this approach is relatively specific for the detection of mutants with insertions near the Mecom locus (also known as Evi1 ) or its close relative Prdm16 , both of which were shown to be clinically relevant as inducers of clonal imbalance in clinical trials for X-CGD [ 55 , 56 ], X-SCID [ 49 ], WAS [ 57 ], it has been accepted by regulatory authorities as part of the pre-clinical safety assessment.…”

“…A more accurate prediction is performed with the SAGA approach that classifies integrating retroviral vectors using machine learning algorithms to detect the activation of gene expression pathways connected to oncogenesis during the course of in vitro cell immortalization [ 58 ]. However, due to the specific culture conditions, both assays present an intrinsic myeloid bias, and thus Bastone and colleagues [ 54 ] have introduced the SAGA-XL assay that follows a similar bioinformatic strategy enabling the identification of lymphoid genotoxicity predictors. Notably, it should be mentioned that the onset of leukemias in HSC gene therapy is not always the result of vector-induced insertional mutagenesis, since in the case of the Lyfgenia™ trials, the leukemic blasts were devoid of vector genetic material, clearly suggesting that the dysplasias were independent of the vector in this specific setting and were rather associated with the overall stemness/fitness of the graft and/or the specific pathophysiology of SCD.…”