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Establishment of CRFK cells for vaccine production by inactivating endogenous retrovirus with TALEN technology

Shimode, Sayumi Sakuma, Tetsushi Yamamoto, Takashi Miyazawa, Takayuki 京都大学 DOI:10.1038/s41598-022-10497-1

2022

概要

Endogenous retroviruses (ERVs) are retroviral sequences present in the host genomes. Although most ERVs are inactivated, some are produced as replication-competent viruses from host cells. We previously reported that several live-attenuated vaccines for companion animals prepared using the Crandell-Rees feline kidney (CRFK) cell line were contaminated with a replication-competent feline ERV termed RD-114 virus. We also found that the infectious RD-114 virus can be generated by recombination between multiple RD-114 virus-related proviruses (RDRSs) in CRFK cells. In this study, we knocked out RDRS env genes using the genome-editing tool TAL Effector Nuclease (TALEN) to reduce the risk of contamination by infectious ERVs in vaccine products. As a result, we succeeded in establishing RDRS knockout CRFK cells (RDKO_CRFK cells) that do not produce infectious RD-114 virus. The growth kinetics of feline herpesvirus type 1, calicivirus, and panleukopenia virus in RDKO_CRFK cells differed from those in parental cells, but all of them showed high titers exceeding 10⁷ TCID₅₀/mL. Infectious RD-114 virus was undetectable in the viral stocks propagated in RDKO_CRFK cells. This study suggested that RDRS env gene-knockout CRFK cells will be useful as a cell line for the manufacture of live-attenuated vaccines or biological substances with no risk of contamination with infectious ERV.

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Acknowledgements

We are grateful to Prof. O. Jarrett (Glasgow University, Glasgow, U.K.) for providing QN10S cells. This work

was supported by JSPS KAKENHI [Grant Numbers JP16K21129, JP20K15692, JP20H03150, and JP22K06043].

Author contributions

S.S. and T.M. designed the experiments. S.S. performed the experiments. T.S. and T.Y. designed and prepared

TALEN constructs. S.S., T.M., T.S., and T.Y. wrote the manuscript. All authors read and approved the final

manuscript.

Competing interests The authors declare no competing interests.

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Additional information

Supplementary Information The online version contains supplementary material available at https://​doi.​org/​

10.​1038/​s41598-​022-​10497-1.

Correspondence and requests for materials should be addressed to T.M.

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