ArticleCytotherapy2023
Reference gene selection for clinical chimeric antigen receptor T-cell product vector copy number assays.
Article in Cytotherapy, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 12 citations in OpenAlex.
- Perspectives on PCR-Based Cellular Kinetics and Biodistribution Analysis for Emerging Adoptive Cell Therapies.The AAPS journal · 2026Review
- Efficient and precise inversion of genomic DNA from large to chromosomal scale.Nature chemical biology · 2026Article
- Multiplexed Digital PCR Reference Gene Measurement for Genomic and Cell-Free DNA Analysis.Cells · 2025Article
- Droplet digital PCR assay for precise determination of FRS2 gene copy number in bladder cancer.BMC cancer · 2025Article
- A method to validate viral copy-number assay involving a hybrid amplicon and duplex droplet digital PCR.Molecular therapy. Methods & clinical development · 2025Article
- New insights on potency assays from recent advances and discoveries in CAR T-cell therapy.Frontiers in immunology · 2025Review
- Harnessing eukaryotic retroelement proteins for transgene insertion into human safe-harbor loci.Nature biotechnology · 2025Article
- Novel liquid biopsy CNV biomarkers in malignant melanoma.Scientific reports · 2024Article
- Clinically accessible amplitude-based multiplex ddPCR assay for tryptase genotyping.Scientific reports · 2024Article
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Authors and funding
12 authors at 2 institutions in 2 countries.
Funding
Abstract
BACKGROUND
aimsReference genes are an essential part of clinical assays such as droplet digital polymerase chain reaction (ddPCR), which measure the number of copies of vector integrated into genetically engineered cells and the loss of plasmids in reprogrammed cells used in clinical cell therapies. Care should be taken to select reference genes, because it has been discovered that there may be thousands of variations in copy number from genomic segments among different individuals. In addition, within the same person in the context of cancer and other proliferative disorders, substantial parts of the genome also can differ in copy number between cells from diseased and healthy people. The purpose of this study was to identify reference genes that could be used for copy number variation analysis of transduced chimeric antigen receptor T cells and for plasmid loss analysis in induced pluripotent stem cells using ddPCR.
methodsWe used The Cancer Genome Atlas (TCGA) to evaluate candidate reference genes. If TCGA found a candidate gene to have low copy number variance in cancer, ddPCR was used to measure the copy numbers of the potential reference gene in cells from healthy subjects, cancer cell lines and patients with acute lymphocytic leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers.
resultsIn addition to the rPP30 gene, which we have has been using in our copy number assays, three other candidate reference genes were evaluated using TCGA, and this analysis found that none of the four gene regions (AGO1, AP3B1, MKL2 and rPP30) were amplified or deleted in all of the cancer cell types that are currently being treated with cellular therapies by our facility. The number of copies of the genes AP3B1, AGO1, rPP30 and MKL2 measured by ddPCR was similar among cells from healthy subjects. We found that AGO1 had copy number alteration in some of the clinical samples, and the number of copies of the genes AP3B1, MKL2 and rPP30 measured by ddPCR was similar among cells from patients with the cancer cell types that are currently being treated with genetically engineered T-cell therapies by our facility.
conclusionsBased on our current results, the three genes, AP3B1, MKL2 and rPP30, are suitable for use as reference genes for assays measuring vector copy number in chimeric antigen receptor T cells produced from patients with acute leukemia, lymphoma, multiple myeloma and human papillomavirus-associated cancers. We will continue to evaluate AGO1 on our future samples.
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