Comparing sequencing methods to detect chimeric sites for ex vivo cell and gene therapy and developing ISAL: A bioinformatics tool to monitor clonal dynamics of chimeric sites.

Abstract

e15204 Background: Ex vivo cell and gene therapies such as autologous CAR-T cells recently have emerged as core modality for targeted gene delivery. Distinctly, allogeneic stem cell-mediated gene therapy provides an advantage of scalable production through long-term expansion; among stem cells, mesenchymal stem cells (MSC) offer additional value as versatile cellular vehicles for their innate homing properties to cancer and injured sites via chemokine signals. Using integrating vectors such as retroviral vectors to introduce stable ex vivo transgene expression raises a concern for insertional mutagenesis. In this study, we have developed ISAL (Integration Site Analysis of LTR), bioinformatics workflow designed to monitor clonal dynamics of chimeric sites, compared performance of sequencing methods in various contexts, and evaluated the long-term clonal diversity and genomic safety of allogeneic MSC transduced with a bacterial suicide gene, cytosine deaminase (CD). Methods: MSC/CD (MSC transduced for CD gene expression) were analyzed at various time points throughout long-term culture. ISAL was used to compare the performance of sequencing methods—whole genome sequencing (WGS), restriction-enzyme mediated amplification, and LTR-specific amplification—to detect integration sites with varying heterogeneity. After identifying the optimal sequencing method, we tracked clonal dynamics and annotated integration sites to assess potential tumorigenicity associated with long-term expansion. Results: LTR-specific amplification was selected for its top performance and in conjunction with ISAL, achieved accuracy of 99% and detection limit of 1% clonal purity, outperforming WGS and restriction-enzyme mediated amplification in heterogeneous contexts. While clonal diversity decreased over long-term culture, genomic profile of insertion sites remained consistent. No emergence of prominent integration sites near tumorigenic regions was observed, suggesting that genomic stability was maintained throughout the long-term expansion. Conclusions: ISAL is an efficient tool for monitoring genomic stability in cell and gene therapy. Our findings suggest that observed clonal reduction may reflect the innate heterogeneity of stem cells and varying stemness potential rather than the emergence of dominant, potentially malignant clones. This bioinformatic profiling underscores the genomic stability of cell and gene therapy including ex vivo CAR-T cells and engineered allogeneic MSC and facilitates their clinical application for gene therapy.