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Updated: Jun 14, 2025
Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells
Published on: February 2, 2013
Assessing a single-cell multi-omic analytic platform to characterize ex vivo-engineered T-cell therapy products
Maryam Moshref1, Jerry Hung-Hao Lo2, Andrew McKay3
1Cell Therapy Engineering and Development, Genentech, South San Francisco, CA, United States.
Abstract:
Genetically engineered CD8+ T cells are being explored for the treatment of various cancers. Analytical characterization represents a major challenge in the development of genetically engineered cell therapies, especially assessing the potential off-target editing and product heterogeneity. As conventional sequencing techniques only provide information at the bulk level, they are unable to detect off-target CRISPR translocation or editing events occurring in minor cell subpopulations. In this study, we report the analytical development of a single-cell multi-omics DNA and protein assay to characterize genetically engineered cell products for safety and genotoxicity assessment. We were able to quantify on-target edits, off-target events, and potential translocations at the targeting loci with per-cell granularity, providing important characterization data of the final cell product. Conclusion: A single-cell multi-omics approach provides the resolution required to understand the composition of cellular products and identify critical quality attributes (CQAs).
Insights
This study introduces a single-cell multi-omics assay for analyzing genetically engineered T cells. This method precisely quantifies edits and potential off-target events, crucial for cancer therapy safety.
Area of Science:
- Cellular and Molecular Biology
- Genomics and Bioinformatics
- Immunotherapy and Cancer Research
Background:
- Genetically engineered CD8+ T cells show promise for cancer treatment.
- Assessing product safety and heterogeneity is challenging for these therapies.
- Current bulk sequencing methods cannot detect rare off-target genetic events.
Purpose of the Study:
- To develop a single-cell multi-omics assay for comprehensive characterization of genetically engineered cell products.
- To enable precise assessment of on-target edits, off-target events, and translocations.
- To enhance safety and genotoxicity evaluations for cell-based therapies.
Main Methods:
- Development of a single-cell multi-omics assay integrating DNA and protein analysis.
- Application of the assay to characterize genetically engineered cell products.
- Quantification of genetic modifications at the single-cell level.
Main Results:
- The assay successfully quantified on-target edits with single-cell resolution.
- Off-target events and potential translocations were identified at the per-cell level.
- Detailed characterization data for the final cell product was generated.
Conclusions:
- A single-cell multi-omics approach offers the necessary resolution for cellular product analysis.
- This method is vital for understanding product composition and identifying critical quality attributes (CQAs).
- The assay advances the safety assessment of genetically engineered cell therapies.
