Mission Bio, the leader in single-cell multiomics, published research in Molecular Therapy Advances showing that its Tapestri® platform can profile vector copy number (VCN), surface protein expression, and vector integrity across roughly 10,000 individual CAR-T cells in a single assay. The advance arrives as the FDA tightens expectations around VCN reporting, exposing the limits of the industry-standard assay, like droplet digital PCR, which delivers only population averages that obscure what’s happening inside individual cells. This limitation matters: a small fraction of cells carrying excessive vector copies can drive serious long-term safety risks, including insertional mutagenesis and clonal hematopoiesis, and bulk methods cannot detect them. The study was conducted in collaboration with City of Hope, Children’s Hospital Colorado, and the University of Colorado Anschutz.

The FDA’s updated guidance is specific in one key respect, stating that average VCN should be calculated relative to the percentage of CAR-positive cells, not total cells, because the latter denominator systematically underestimates true vector integration rates. This distinction matters because viral vectors integrate into host genomes with high cell-to-cell variability, and the engineered cell, not the bulk population, is the fundamental therapeutic unit. Meeting this standard requires assays that can directly measure integration events in individual transduced cells rather than inferring them from population averages.

Mission Bio’s Tapestri single-cell platform demonstrated in this study the measurement of VCN, surface protein expression, and vector integrity across ~10000 individual cells simultaneously. Based on validation studies across different vectors, the single-cell transduction assay demonstrated 99.2% to 99.9% sensitivity and 98.8% to 99.6% specificity.

Evaluating VCN at the single-cell level fundamentally improves the characterization of CAR-T therapies by linking genotypic data directly to phenotypic protein markers. Utilizing a 45-plex oligo-conjugated antibody panel, the workflow quantitatively measures surface protein expression for precise lineage assignment alongside VCN. Key technical findings from the validation study include:

• Proactive Detection of Safety Risks: Despite a low average VCN, the Tapestri single-cell multiomic platform successfully identified that approximately 2% of cells harbored five or more vector copies. Detecting these rare, high-VCN cells is critical, as excessive vector copies pose a recognized safety risk for insertional mutagenesis and clonal hematopoiesis phenotypes.
• Lineage-Specific Integration Bias: When analyzing a bi-cistronic CD19xCD22 CAR-T product, single-cell profiling revealed significant differences in transduction percentage and average VCN across T-cell lineages. CD4+ T cells exhibited an average VCN of approximately 1.06, while CD8+ T cells demonstrated an average VCN of roughly 0.34. This difference was driven by both a lower transduction rate in CD8+ cells and a bias toward higher VCNs within the CD4+ transduced subset. These findings serve as a proof-of-concept for how single-cell resolution can distinguish global transduction trends from lineage-specific integration patterns that bulk methods cannot resolve.
• Confirming Stable Manufacturing Processes: In a developmental batch of CAR-T cells cultured for seven days post-transduction, the overall VCN distribution closely aligned with a theoretical Poisson distribution (μ=1.62, R²=0.98). This alignment suggests the manufacturing process did not indicate significant VCN-dependent clonal expansion during this timeframe. This demonstrates how a robust single-cell multiomic assay can provide value in the manufacturing and QC processes for cell therapies.
• Monitoring Vector Integrity: Leveraging a next-generation sequencing (NGS) readout, the bioinformatics pipeline surveyed vector integrity at single-nucleotide resolution. The single-cell multiomic assay successfully identified a spontaneous mutation occurring at vector position 408:G/A, demonstrating the capability to detect vector sequence variation, including SNVs, truncations, and indels that critically impact the genomic integrity and safety profile of a cell therapy product.

Bulk VCN measurement was never wrong, but it was incomplete. The cells driving safety risk often aren’t the ones driving the average, and single-cell resolution is what tells them apart,” said Shu Wang, Ph.D., Executive Director, Bioinformatics at Mission Bio. “That’s the level of detail regulators are moving toward, and the level of detail developers need to de-risk their programs.

Developers ready to apply single-cell VCN profiling to their own programs can work with Mission Bio’s Assay Services for custom VCN amplicon panel design and complex single-cell data analysis. Once the single-cell VCN assay is optimized for a specific CAR construct, the complete methodology can be seamlessly transferred to the investigator’s or developer’s laboratory to establish routine VCN measurement for preclinical discovery, process development, and rigorous quality control.

Mission Bio is the single-cell multi-omics leader. The company’s Tapestri Platform is unique in its capabilities, offering an unparalleled level of granularity and precision that is critical for complex research areas such as cancer studies, pharmaceutical development, and advanced cell and gene therapies. Unlike traditional standard of care methods, Tapestri provides a level of precision that opens the door for more tailored and effective treatment strategies. Researchers globally depend on Tapestri to identify rare cell populations, understand mechanisms of therapeutic resistance and response, and establish key quality metrics for next-generation medical treatments. With the Tapestri Platform, Mission Bio continues to set the standard in the field, contributing significantly to the progress of personalized medicine and targeted therapies. To learn more about Mission Bio and the Tapestri Platform, please visit missionbio.com.