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Combining assays to evaluate genotoxicity risk of integrating vectors

VP, Toxicology, Research and Preclinical Development

Richard W. Pfeifer, Ph.D., DABT | VP, Toxicology, Research and Preclinical Development

To continue evolving gene therapy, many in our industry are working to characterize and better understand safety concerns. Data on research attempting to do just this, and supported by AVROBIO (my employer), was presented at the European Society of Gene and Cell Therapy (ESGCT) in Scotland on Oct. 14, 2022, by my esteemed colleague Holly Horton, Ph.D., senior director Toxicology & Preclinical Development at AVROBIO.

A vector comprises multiple elements designed to provide the therapeutic gene that is integrated into a patient’s hematopoietic stem cells (HSCs). One key element in a vector is the promotor, which influences how strongly a gene is expressed. Strong gene expression can be critical for sufficient efficacy, but vectors with a powerful promoter are at risk of activating genes close to the integration site, which can lead to safety concerns. It is important to try to reduce the risk of activating proto-oncogenes, or genes that may lead to cancer.

In collaboration with Professor Axel Schambach, Ph.D., Institute of Experimental Hematology, Hannover Medical School, Germany, AVROBIO is researching two preclinical cell-based assays — in vitro immortalization (IVIM) assay and the novel surrogate assay for genotoxicity assessment (SAGA) — to evaluate viral vectors in a preclinical setting. Together these assays are designed to assess a vector’s likelihood to exhibit genotoxic behavior and to monitor if these vectors activate proto-oncogenes. The Hannover Medical School team working with Professor Schambach and Michael Rothe, Ph.D., has previously published its data in Molecular Therapy.

The IVIM assay is designed to estimate the risk of vector-induced cellular transformation. The technique assesses genotoxicity by determining how likely a vector is to insert near and activate proto-oncogenes and lead to an over-proliferation of cells. To evaluate the risk of vector-induced cellular transformation, mouse hematopoietic and stem progenitor cells (HSPCs) were transduced with a panel of vectors and resulting cell growth was compared to a non-transforming mock control and a positive mutagenic control.

The newer SAGA assay assesses genotoxicity more directly. SAGA relies on the observation that genotoxic vectors induce a unique gene expression signature that has been linked to stemness and oncogenesis in mouse HSPCs. Machine learning algorithms developed from transcriptional data of known genotoxic vectors are used to estimate the transformational potential of candidate vectors. On a set of benchmark vectors with known genotoxic potential, the SAGA assay achieved an accuracy of 90.9%, making it more sensitive than previous assays.

In the research presented at ESGCT, the IVIM and SAGA assays were used to evaluate the mutagenic and genotoxic risk potential of lentiviral vectors containing two promoters – the EF1 α short promoter (EFS) and the murine myeloproliferative sarcoma virus promoter (MND). Growth of cells transduced with vectors containing the EFS promoter was statistically different from the mutagenic positive control, whereas growth of cells with the MND promoter was not significantly different from the positive control, suggesting the potential for insertional transformation. Similarly, SAGA analysis of these vectors found the EFS vectors had low normalized enrichment scores of < 1, indicating low mutagenic risk, whereas the MND vector showed a gene enrichment score associated with insertional oncogenesis.

By continually looking at research designed to evaluate and understand the vectors used in gene therapy and using assays in the development process to better assess vector behavior, we have the potential to pave the way for safer gene therapy vectors used in clinical trials.