{"title":"Accurate vector copy number determination in gammaretroviral vector producer cell clones using triplex digital droplet PCR","authors":"Tomomine Iida , Yoshiki Nakamura , Katsuhiko Yamamoto , Eiki Maeda , Yukihiro Ikeda","doi":"10.1016/j.jviromet.2024.115075","DOIUrl":null,"url":null,"abstract":"<div><div>Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer– probe set targeting the packaging signal sequence common to murine leukemia virus– and murine stem cell virus– based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115075"},"PeriodicalIF":2.2000,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of virological methods","FirstCategoryId":"3","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0166093424002003","RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
Abstract
Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer– probe set targeting the packaging signal sequence common to murine leukemia virus– and murine stem cell virus– based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients.
期刊介绍:
The Journal of Virological Methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery.
The methods may include, but not limited to, the study of:
Viral components and morphology-
Virus isolation, propagation and development of viral vectors-
Viral pathogenesis, oncogenesis, vaccines and antivirals-
Virus replication, host-pathogen interactions and responses-
Virus transmission, prevention, control and treatment-
Viral metagenomics and virome-
Virus ecology, adaption and evolution-
Applied virology such as nanotechnology-
Viral diagnosis with novelty and comprehensive evaluation.
We seek articles, systematic reviews, meta-analyses and laboratory protocols that include comprehensive technical details with statistical confirmations that provide validations against current best practice, international standards or quality assurance programs and which advance knowledge in virology leading to improved medical, veterinary or agricultural practices and management.