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

Abstract

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.

Multiplex secretome engineering enhances recombinant protein production and purity

“…The dramatically lowered HCP ppm values during purification of a mAb will facilitate downstream processes. These HCP-reduced knockouts can be combined with additional advantageous genetic modifications, such as the production of glycoengineered proteins [15][16][17][18] , higher viability during long culture times 19 , viral resistance or elimination [20][21][22] , and/or clones with higher protein production stability of mAbs 23 , to create predictable upstream and downstream processes with full control over critical process parameters.…”

Preclinical evaluation of manufacturable SARS-CoV-2 spike virus-like particles produced in Chinese Hamster Ovary cells

“…As shown in Fig. 2a , CHO-C2 gives S-VLP yields similar to non-engineered cells but without the presence ERVLPs, as indicated by Gag western blotting (Gag is the principal protein component responsible for the formation and budding of these particles) 18 . Volumetric productivity for S-VLPs produced in CHO-C2 cells (harvested at 5 days post-transfection) averaged 8.5 ± 0.4 mg/L (standard error of the mean [SEM]; n = 6), based on S protein content of iodixanol-sedimented particles (Fig.…”

“…The release of ERVLPs by CHO cells 29 , 30 could be a concern for recombinant enveloped VLP vaccine manufacturing, in particular, because of the likely structural and biophysical similarity of recombinant enveloped VLPs and endogenous CHO ERVLPs that would make separation of the two entities challenging during downstream processing steps. CRISPR-Cas9 has been used to target retrovirus-like proviral elements like gag and env in the CHO genome 18 , and we used this approach similarly to engineer a CHO cell line, CHO-C2, with disrupted ERVLP production. As shown in Fig.…”

“…Clone CHO-C2 was derived from the parental CHO 2353 by targeting and disrupting retroviral gag and env sequences responsible for ERVLP formation and release using CRISPR-Cas9 technology similarly as described in ref. 18 . Cells were maintained in a proprietary media formulation in polycarbonate Erlenmeyer flasks with 0.2 µm vent cap (Corning) with constant orbital shaking at 120 rpm in a humidified incubator with 5% CO 2 at 37 °C.…”

CRISPR Technologies in Chinese Hamster Ovary Cell Line Engineering

“…This review sums up the CRISPR-Cas9 system applications in CHO cell line engineering (Figure 1). [17][18][19][20][21][22][23]; (B) targeted gene knockin [24][25][26][27]; (C) CRISPR activation [28][29][30]; (D) CRISPR interference [31][32][33]; (E) DNA base editing [34]; (F) targeted mRNA knockdown [35,36].…”