Multicopy Targeted Integration for Accelerated Development of High-Producing Chinese Hamster Ovary Cells

Abstract

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An optimized genome-wide, virus-free CRISPR screen for mammalian cells

“…Resistance to TM was likewise observed in these three Zfx KO cell lines ( Figure 7G ). TM resistance obtained from Zfx KO was also observed in two established Enbrel-producing CHO cell lines ( Sergeeva et al, 2020 ) (dual-RMCE ETN2_T9) with Zfx KO ( Figure 7H ). Thus, the VF CRISPR screen can successfully identify candidate genes via gRNA enrichment analysis.…”

Rational design and construction of multi-copy biomanufacturing islands in mammalian cells

“…Thus far, work has primarily focused on the identification of transcriptional hotspots in the host cell line's genome (mainly CHO cells) and the establishment of genetic platforms for the targeted integration of transgenes (see ( 58 ) for a review). Despite the central role occupied by gene amplification in the current cell line development scheme, more limited has been the progress in the direction of programmed transgene dosage increase ( 26 , 27 ), possibly owing to the difficulties connected with handling large amounts of repetitive sequences in the form of repeated gene expression cassettes. The main objective of this work is to provide a new strategy for achieving controlled transgene dosage increase with the goal of rationally designing, from the bottom up, genomic islands for bioproduction whose territory is fully mapped, in contrast to the current cell line development landscape where neither the integration sites nor transgene copy number are defined, even in the producer clones that are ultimately selected for a production project.…”

“…While site-specific integration platforms have already found applications in industry in connection with the accelerated development of stable producer cell lines for the early molecular assessment of candidate protein therapeutics, product titers can seldom match those obtained with the best clones derived from a random integration and selection approach, where multiple GOI copies are normally integrated and active in the host genome ( 21 ). With the objective of narrowing the gap in product titers between the random and targeted integration platforms, there have been attempts at increasing gene dosage by simultaneously targeting more than one genomic site ( 22 ), by concatenating multiple GOI copies ( 23–25 ), or through a combination of both approaches, that is, targeting multiple GOI copies to two or more loci ( 26 , 27 ). Despite the promise held by these approaches, they are limited with respect to scalability, as evidenced by a recent study reporting GOI loss and, concomitantly, a lack of product increase when four or more GOI-bearing, identical gene expression cassettes were integrated at a single genomic site, potentially owing to recombination between repeated sequences ( 27 ).…”

“…With the objective of narrowing the gap in product titers between the random and targeted integration platforms, there have been attempts at increasing gene dosage by simultaneously targeting more than one genomic site ( 22 ), by concatenating multiple GOI copies ( 23–25 ), or through a combination of both approaches, that is, targeting multiple GOI copies to two or more loci ( 26 , 27 ). Despite the promise held by these approaches, they are limited with respect to scalability, as evidenced by a recent study reporting GOI loss and, concomitantly, a lack of product increase when four or more GOI-bearing, identical gene expression cassettes were integrated at a single genomic site, potentially owing to recombination between repeated sequences ( 27 ).…”

Development of a targeted integration Chinese hamster ovary host directly targeting either one or two vectors simultaneously to a single locus using the Cre/Lox recombinase‐mediated cassette exchange system

“…More recently, a TI system targeting multicopies at two genomic sites has been developed as well. 30 Our two-plasmid based RMCE approach provides a unique way of potentially targeting twice as many copies of HC and LC to a single locus compared with the conventional single-plasmid based RMCE. The expanded targeting capacity not only increases the flexibility of modulating HC and LC ratios of standard and multichained complex antibodies to improve productivity, but also permits the co-expression of transgenes, endogenous genes or shRNA with the antibodies to modify cellular pathways.…”