Eukaryotic cells for protein manufacturing and methods of making them
Inventors
Mermod, Nicolas • Duroy, Pierre-Olivier • Bosshard, Sandra • Le Mercier, Philippe
Assignees
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Publication Number
US-11739351-B2
Publication Date
2023-08-29
Expiration Date
Abstract
Disclosed are mammalian cells and mammalian cell lines that have a reduced load of remnants of past viral/retroviral infections and methods of producing and using the same.
Core Innovation
The invention relates to engineered CHO (Chinese Hamster Ovary) cells whose genomes comprise endogenous retrovirus (ERV) elements. The ERV elements include sequences encoding a Gag protein, and the Gag-encoding sequences comprise SEQ ID No: 30, or a sequence having more than 90% sequence identity with SEQ ID No: 30, altered so that the ERV elements do not encode a functional Gag protein.
The disclosed approach addresses expressed gammaretroviral ERV elements in CHO-M/SURE CHO-M cells and aims to reduce expression and release of endogenous retrovirus remnants. The described framework identifies expressed gammaretroviral ERV elements, including KoRV, MMTV, and MLV, and characterizes them using sequencing and sequence comparison, methylation/CpG analyses, and RNA/cDNA expression analyses.
The disclosed ERV-inactivation strategy includes genome-editing directed to key Gag motifs, including PPYP/PPXY-related motifs and myristoylation motifs, and combines this with modulation of recombination pathways to favor deletions or altered outcomes at the GAG coding sequence. The document describes validation outcomes showing reduction or near disappearance of Gag mRNA and high mutation of GAG coding sequences, consistent with ERV elements not encoding a functional Gag protein.
Claims Coverage
The independent claim covers an engineered CHO cell with ERV elements containing a Gag-encoding sequence (SEQ ID No: 30 or a >90% sequence-identity variant) that is altered so the ERV elements do not encode a functional Gag protein. Dependent claim refinements add quantitative constraints on ERV alteration coverage and release suppression, define aspects of the altered sequence changes, and specify example marker and targeting-related features.
Engineered CHO cell with altered SEQ ID No: 30 Gag-encoding ERV sequence
An engineered CHO cell comprising a genome of the CHO cell, wherein the genome comprises endogenous retrovirus (ERV) elements comprising sequences encoding a Gag protein wherein the sequences encoding the Gag protein comprise SEQ ID No: 30, or a sequence having more than 90% sequence identity with SEQ ID No: 30, wherein the SEQ ID No: 30 or the sequence having more than 90% sequence identity with SEQ ID No: 30 is altered to comprise at least one deletion, at least one addition, at least one substitution or combinations thereof resulting in an altered sequence, wherein the ERV elements comprising the altered sequence do not encode a functional Gag protein.
Suppression or elimination of ERV release for specified gammaretroviral ERVs
The engineered cell wherein the ERV elements comprise altered sequences adapted to suppress or eliminate release of specified gammaretroviral ERVs by providing suppression or elimination of greater than 60%, 70%, 80%, 90%, 95%, or 100%.
Altered sequence coverage across ERV elements
The engineered cell wherein ERV elements in the genome have an altered sequence comprising at least a specified percentage, up to all of the ERV elements present.
Size constraints on deletion, addition, and substitution in nucleic acids
The engineered cell wherein deletions, additions, or substitutions include more than a specified number of nucleic acids.
Targeted integration for altering SEQ ID No: 30
The engineered cell wherein SEQ ID No: 30, or a sequence with more than 90% identity to SEQ ID No: 30, is altered via a targeted integration addition.
Marker protein specified as GFP
The engineered cell using GFP (green fluorescent protein) as the marker protein.
Across the claim set, the core coverage is directed to engineered CHO cells in which ERV elements containing a SEQ ID No: 30-based Gag-encoding sequence are altered by deletion, addition, or substitution so that the ERV elements do not encode a functional Gag protein. Dependent claims further narrow the scope by specifying gammaretroviral ERV release suppression or elimination levels, the fraction of ERV elements carrying the altered sequence, quantitative constraints on change size, and example features including targeted integration additions and a GFP marker protein.
Stated Advantages
Reduces expression and release of endogenous retrovirus remnants by altering ERV Gag sequences so ERV elements do not encode a functional Gag protein.
Achieves suppression or elimination of ERV release for specified gammaretroviral ERVs at specified proportions greater than 60% up to 100%.
Provides ERV inactivation characterized by reduction or near disappearance of Gag mRNA and high mutation of GAG coding sequences.
Documented Applications
Engineered CHO protein-manufacturing cell lines with reduced expression and release of endogenous retrovirus (ERV) remnants, including regulatory characterization and detection of adventitious viral-like agents in the context of mammalian cell manufacturing.
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