Measuring the Mosaicism of Genome Integrity and Toxicity Events Associated with Gene and Cell Therapies at a Single-Cell Level

Recent advancements in cell and gene therapy are transforming the treatment landscape for diseases like cancer and rare genetic disorders. Precision gene therapies targeting conditions such as sickle cell disease, beta-thalassemia, and B-cell malignancies have shown remarkable efficacy in correcting genetic defects at their source, paving the way for long-term cures.

Despite these breakthroughs, the gene editing process can result in heterogeneous cell populations, some of which may carry undesirable outcomes that pose risks of genomic toxicity and potential malignancies. These adverse effects may include off-target edits, copy number variations, and chromosomal aberrations such as translocations or large indels. Thus, the success of gene therapies hinges on the ability to accurately measure and understand these events. Given that "cells" are the fundamental units of gene editing products, it is crucial to assess the co-occurrence of editing outcomes and potential genotoxic events at the single-cell level.

In this study, we introduce an advanced single-cell technology that integrates microfluidics and multiplex PCR to concurrently measure several critical aspects of gene editing. This assay evaluates the co-occurrence and zygosity of on-target and off-target edits, quantitatively detects translocations between predicted edit sites, and maps the genomic copy number variation (CNV) landscape, analyzing over 10,000 cells in parallel. With single-cell resolution, this method provides a detailed and comprehensive view of the heterogeneous editing profiles in gene-edited products. It enables precise and rapid assessment of editing outcomes and overall genomic integrity, facilitating the identification of potential malignancies and enhancing the safety and efficacy of gene therapies.

To validate the robustness of this workflow, we employed various orthogonally validated samples with different levels of adverse effects from multiple gene editing experiments. Using a targeted sequencing panel, we measured on- and off-target editing activity, detected structural variations, and assessed genome-wide CNVs at the single-cell level in a single assay. The resulting data were analyzed with a novel bioinformatics pipeline specifically designed to accurately measure all these modalities simultaneously. Additionally, for locus-level CNV detection, we demonstrated the performance of the single-cell CNV assay at a focal level. This was exemplified by confirming a Chr20q copy number alteration in PGP-1 iPSC cell lines, which conventional methods such as G-banding and CMA failed to detect.

This high-throughput single-cell NGS assay marks a significant advancement in gene editing analysis by effectively capturing both intended and unintended consequences of genome editing at various resolutions. Its dual functionality not only enhances the optimization of gene editing protocols but also provides a comprehensive and efficient approach for evaluating the safety aspects of gene-modified therapies. By enabling detailed assessments of genomic integrity and potential malignancies, this method significantly contributes to key CQA that could guide the development of safer and more effective gene therapies.