Characterization and mutagenesis of Chinese hamster ovary cells endogenous retroviruses to inactivate viral particle release

The Chinese hamster ovary (CHO) cells used to produce biopharmaceutical
proteins are known to contain type‐C endogenous retrovirus (ERV) sequences
in their genome and to release retroviral‐like particles. Although
evidence for their infectivity is missing, this has raised safety
concerns. As the genomic origin of these particles remained unclear, we
characterized type‐C ERV elements at the genome, transcriptome, and viral
particle RNA levels. We identified 173 type‐C ERV sequences clustering
into three functionally conserved groups. Transcripts from one type‐C ERV
group were full‐ length, with intact open reading frames, and cognate
viral genome RNA was loaded into retroviral‐like particles, suggesting
that this ERV group may produce functional viruses. CRISPR‐Cas9 genome
editing was used to disrupt the gag gene of the expressed type‐C ERV
group. Comparison of CRISPR‐derived mutations at the DNA and RNA level led
to the identification of a single ERV as the main source of the release of
RNA‐loaded viral particles. Clones bearing a Gag loss‐of‐function mutation
in this ERV showed a reduction of RNA‐containing viral particle release
down to detection limits, without compromising cell growth or therapeutic
protein production. Overall, our study provides a strategy to mitigate
potential viral particle contaminations resulting from ERVs during
biopharmaceutical manufacturing.