Characterization and inactivation of endogenous retroviruses in chinese hamster ovary cells

Inventors

Duroy, Pierre-Olivier • Bosshard, Sandra • Le Mercier, Philippe • Schmid-Siegert, Emanuel • Mermod, Nicolas

Assignees

Selexs SA • Selexis SA

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.

Core Innovation

The invention characterizes Chinese hamster ovary (CHO) endogenous Type-C endogenous retroviruses (ERVs) in CHO-K1 and identifies a transcribed group 1 gag-pol-env locus whose viral RNA is loaded into retroviral-like particles. The identified locus produces viral-like particles (VLPs), viral particles (VPs), and retroviral-like particles (RVP/RVLP) in connection with ERV expression and viral RNA incorporation.

The invention addresses viral contamination from CHO-derived retroviral-like particles by disrupting conserved gag budding motifs. Specifically, disrupting conserved gag budding motifs within gag, including an N-terminal Myr motif and a PPYP (PPxY) motif, enables identification of a single ERV locus responsible for particle release.

With the identified locus, introducing gag loss-of-function mutations into the single ERV locus reduces viral RNA-loaded particle output by greater than 250-fold. The invention further states that ERV mutagenesis does not significantly impair CHO cell growth, cell size, or recombinant therapeutic IgG production, supporting a strategy to mitigate CHO-derived viral contamination during biopharmaceutical manufacturing.

Claims Coverage

The independent claim is directed to an engineered cell having a genome engineered to include integrated ERV sequences including at least one full-length ERV sequence corresponding to SEQ ID NO: 3 or having 95% sequence identity. The claim structure requires that one to twenty alterations are within one or more gag sequences, and that at least one alteration is within a gag gene of the at least one full-length ERV sequence resulting in at least one altered full-length ERV sequence.

Overall claim coverage focuses on engineered cells with integrated ERV sequences containing a full-length ERV corresponding to SEQ ID NO: 3, where edits are confined to the gag sequences and include gag gene alterations that yield altered full-length ERV sequences. Dependent claim refinements further narrow which gag sequences are altered, the types and scopes of alterations, total alteration count, and viral release outcomes such as no or substantially no RVP.

Stated Advantages

Documented Applications