CHO Cell ERV Gag Editing to Reduce Retroviral Particle Release
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Solution Overview
Problem
Chinese hamster ovary (CHO) cells used in biopharmaceutical production pose a risk of viral contamination due to the presence of endogenous retroviruses, particularly type-C ERVs, which can potentially produce viral-like particles (VLPs) that interfere with detection of other adventitious agents and pose a transmission risk to humans.
Innovation Solution
In-depth characterization of type-C ERV sequences in CHO cells reveals that mutating the gag gene of these sequences using CRISPR-Cas9 technology, specifically introducing loss-of-function mutations, significantly reduces the release of functional viral particles by more than 250-fold.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CHO cells are used as expression system, then productivity of biopharmaceutical products is improved, but risk of viral contamination by endogenous retroviruses increases
Solution Approach 1:
The invention extracts and removes the harmful endogenous retrovirus sequences from the CHO cell genome using CRISPR-Cas9 genome editing technology. Specific guide RNAs target the ERV sequences for cleavage, and the cellular repair mechanisms introduce mutations that inactivate the retroviral elements, thereby eliminating the source of viral contamination while preserving the cell line's productivity
Solution Approach 2:
The invention converts the harmful effect of endogenous retroviruses into a benefit by using their own genetic sequences as targets for CRISPR-Cas9 editing. The guide RNAs are designed to complement ERV sequences, directing the Cas9 nuclease to precisely locate and mutate these elements, transforming the potential contamination risk into a controlled editing opportunity that enhances safety
2Quantity of substance
If endogenous retrovirus sequences are present in CHO cells, then viral-like particles are produced, but sensitivity of detection of other adventitious agents is reduced
Solution Approach 1:
The invention removes the source of viral-like particles by targeting and mutating the endogenous retrovirus sequences in the CHO genome. CRISPR-Cas9 mediated editing introduces frameshift mutations and premature stop codons in the gag, pol, and env genes, preventing VLP production and eliminating interference with adventitious agent detection assays
3Reliability
If CRISPR-Cas9 is used to mutate gag gene, then release of functional viral particles is reduced by more than 250-fold, but complexity of genome editing process increases
Solution Approach 1:
The invention segments the complex genome editing task into manageable components: design of specific guide RNAs for each ERV locus, selection of optimal target sites within gag/pol/env genes, and modular delivery of CRISPR components. This segmentation makes the complex process of mutating multiple ERV sequences systematic and controllable
Solution Approach 2:
The invention changes key parameters of the ERV sequences by introducing specific mutations (frameshifts, stop codons) at targeted locations. By modifying the genetic code parameters of the gag, pol, and env genes, the CRISPR-Cas9 system permanently alters the retroviral sequences to prevent functional particle production while maintaining genomic stability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The engineered CHO cells with targeted mutations in the gag gene effectively minimize the release of viral particles, enhancing safety and maintaining transgene product production, thereby reducing the risk of viral contamination and improving the safety profile of biopharmaceutical production.
Implementation Method 1
mutating the gag gene of these sequences using CRISPR-Cas9 technology, specifically introducing loss-of-function mutations
Data Source
AI summary
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.