Type-C ERV Characterization and Gag Inactivation in CHO Cells
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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 infectious viral particles, complicating safety and regulatory compliance.
Innovation Solution
CRISPR-Cas9 genome editing is employed to introduce targeted mutations in the gag gene of type-C ERV sequences, reducing the release of functional viral particles by more than 250-fold, while maintaining transgene product production and minimizing genetic instability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If CRISPR-Cas9 genome editing is used to inactivate endogenous retroviruses in CHO cells, then viral particle release is reduced by more than 250-fold, but the complexity of the manufacturing process increases
Solution Approach 1:
The patent extracts and removes the harmful endogenous retrovirus sequences from the CHO cell genome using CRISPR-Cas9 genome editing. By specifically targeting and excising the ERV sequences, the patent eliminates the source of viral particle production while preserving the rest of the cell genome and its productive functions.
Solution Approach 2:
The patent converts the harmful endogenous retrovirus sequences into a benefit by using them as specific targets for CRISPR-Cas9 mediation. The presence of these ERV sequences, while originally harmful, enables precise genome editing to occur, ultimately eliminating the viral threat and creating safer cell lines for biopharmaceutical production.
2Reliability
If multiple ERV sequences are targeted for inactivation, then viral safety is improved, but the risk of genomic rearrangements and cell death increases
Solution Approach 1:
The patent applies partial action by using a multiplex CRISPR-Cas9 system that can target multiple ERV sequences simultaneously, but only inactivates the specific harmful sequences that are successfully edited. This approach provides viral safety through multiple targets while allowing the cell to survive with partial inactivation, avoiding the need for complete elimination of all potential ERV sites.
Solution Approach 2:
The patent employs beforehand cushioning by designing the CRISPR-Cas9 system with multiple guide RNAs targeting different ERV sequences, creating a buffer against genomic instability. If one editing event causes rearrangement or cell death, other targeted sequences remain as backup targets, and the cell can still achieve viral inactivation through the remaining successful edits.
3Productivity
If CHO cells are used for biopharmaceutical production, then production capacity is maintained, but the risk of hamster to human ERV transmission remains
Solution Approach 1:
The patent extracts and removes the harmful ERV sequences from the CHO cell genome while preserving the cell's productive capabilities for transgene expression. By specifically eliminating only the viral sequences and leaving the rest of the genome intact, the patent maintains production capacity while eliminating transmission risk.
Solution Approach 2:
The patent introduces CRISPR-Cas9 technology as an intermediary tool to mediate between the need for CHO cell productivity and the need to eliminate ERV transmission risk. This intermediary genome editing system enables selective removal of harmful sequences without compromising the cell's biopharmaceutical production capabilities.
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 significantly reduce the release of viral particles, enhancing safety and regulatory compliance by effectively inactivating endogenous retroviruses, thereby ensuring the integrity of biopharmaceutical production.
Implementation Method 1
CRISPR-Cas9 genome editing is employed to introduce targeted mutations in the gag gene of type-C ERV sequences
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.