Type-C endogenous retroviruses (ERVs) embedded in
Chinese hamster ovary (CHO) cells were altered to modify the release of retroviral and / or retroviral-like particles in the culture supernatant. Although evidence for the
infectivity of these particles is missing. their presence has raised safety concerns. 173 type-C ERV sequences that clustered into functionally conserved groups were identified. Transcripts from one type-C ERV group were identified to be full-length with intact open reading frames, and to have corresponding
viral RNA genomes that were loaded into retroviral-like particles. Also,
sequence analysis of the
genomic RNA from viral particles indicated that they may result from few expressed ERV sequences. Disclosed herein is the disruption / alteration of the gag
gene of the expressed ERV group using
CRISPR-Cas9
genome editing. Comparison of
CRISPR-derived mutations at the
DNA and
mRNA level led to the identification of a single ERV locus responsible for the release of
viral RNA-loaded particles from CHO cells. Clones bearing a Gag loss-of-function
mutation in this particular ERV locus showed a reduction of
viral RNA-containing particles in the
cell supernatant by over 250-fold. Notably, ERV
mutagenesis did not compromise
cell growth,
cell size or
recombinant protein production. Provided herein is a new strategy and cells, in particular engineered CHO cells, to mitigate potential contaminations from CHO endogenous retroviruses during
biopharmaceutical manufacturing.