Characterization and inactivation of endogenous retroviruses in chinese hamster ovary cells

Abstract

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.

Description

Background technique [0001] Contamination of biopharmaceutical products by exogenous agents such as viruses can disrupt drug supply, jeopardizing patient safety. Although viral contamination of biopharmaceuticals is rare, they still occur (1), and reducing the risk of viral contamination in therapeutic protein formulations remains a high priority. [0002] Chinese hamster ovary (CHO) cells are the most widely used mammalian expression system for biopharmaceutical products. Among them, CHO cells are the preferred production host due to their superior safety compared to other cell lines used to produce recombinant proteins. For example, studies have shown that CHO cells have reduced susceptibility to certain viral infections (1), including resistance to infections caused by many human and murine retroviruses, some of which are known to infect Other mammalian cells (2, 3). Furthermore, unlike other rodent cells, CHO cells do not appear to produce infectious retroviruses that c...

AI Technical Summary

Problems solved by technology

However, the incomplete characterization of C-type ERV sequences in CHO cells and the lack of a clear link between genomic C-type ERV sequences and viral particles have hindered the establishment of similar ERV inactivation strategies in CHO cells

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