Accurate vector copy number determination in gammaretroviral vector producer cell clones using triplex digital droplet PCR.

Autor: Iida T; Cell Therapy Sciences, Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan; Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan. Electronic address: tomomine.iida@takeda.com., Nakamura Y; Cell Therapy Sciences, Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address: yoshiki.nakamura@takeda.com., Yamamoto K; Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Quality Control, Global Quality, Hikari Plant, Takeda Pharmaceutical Company Limited, 4720, Takeda, Mitsui, Hikari, Yamaguchi 743-8502, Japan. Electronic address: katsuhiko.yamamoto@takeda.com., Maeda E; Cell Therapy Sciences, Research, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address: eiki.maeda@takeda.com., Ikeda Y; Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan; Analytical Development, Pharmaceutical Sciences, Takeda Pharmaceutical Company Limited, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555, Japan. Electronic address: yukihiro.ikeda@takeda.com.
Jazyk: angličtina
Zdroj: Journal of virological methods [J Virol Methods] 2024 Nov 19; Vol. 332, pp. 115075. Date of Electronic Publication: 2024 Nov 19.
DOI: 10.1016/j.jviromet.2024.115075
Abstrakt: 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.
Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The authors are employees of Takeda Pharmaceutical Company Limited, and have no commercial, proprietary, or financial interests in the products or companies described in this article.
(Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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