MAR‐Mediated transgene integration into permissive chromatin and increased expression by recombination pathway engineering

Abstract

Untargeted plasmid integration into mammalian cell genomes remains a poorly understood and inefficient process. The formation of plasmid concatemers and their genomic integration has been ascribed either to non‐homologous end‐joining (NHEJ) or homologous recombination (HR) DNA repair pathways. However, a direct involvement of these pathways has remained unclear. Here, we show that the silencing of many HR factors enhanced plasmid concatemer formation and stable expression of the gene of interest in Chinese hamster ovary (CHO) cells, while the inhibition of NHEJ had no effect. However, genomic integration was decreased by the silencing of specific HR components, such as Rad51, and DNA synthesis‐dependent microhomology‐mediated end‐joining (SD‐MMEJ) activities. Genome‐wide analysis of the integration loci and junction sequences validated the prevalent use of the SD‐MMEJ pathway for transgene integration close to cellular genes, an effect shared with matrix attachment region (MAR) DNA elements that stimulate plasmid integration and expression. Overall, we conclude that SD‐MMEJ is the main mechanism driving the illegitimate genomic integration of foreign DNA in CHO cells, and we provide a recombination engineering approach that increases transgene integration and recombinant protein expression in these cells. Biotechnol. Bioeng. 2017;114: 384–396. © 2016 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals, Inc.

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

“…C‐NHEJ typically leads to small insertion and deletions, whereas alternative end‐joining (alt‐EJ) repair activities relying on microhomologies at the DSB site to anneal broken ends often results in larger and more complex mutations. Although alt‐EJ repair is considered to be a backup pathway in most mammalian cells, we detected between 25% and 55% alt‐EJ compatible junctions when targeting the gag gene, supporting our previous conclusions of intrinsically elevated alt‐EJ activities in CHO cells (Bosshard, Duroy, & Mermod, ; Kostyrko & Mermod, ; Kostyrko et al, ). Interestingly, approximately 10% of all analyzed repair junctions contained insertions templated from other ERV loci, or from the same ERV locus but using a distant sequence, while others manifested apparent duplications devoid of microhomologies, as required by alt‐EJ mechanisms.…”

A chimeric HS4 insulator–scaffold attachment region enhances transgene expression in transfected Chinese hamster ovary cells

“…Matrix attachment regions can increase expression levels of the transgene in stably transfected CHO cells [6][7][8][9][10][11][12]. However, the characteristics and mechanism of MARs function have not been elucidated, and further studies are needed to develop improved methods for transgene expression.…”

“…Moreover, gene copy numbers are related to transgene expression , and the methylation of DNA may reduce transgene expression . Some reports have demonstrated that MARs improve the expression of transgenes through the recombination pathway of synthesis‐dependent microhomology‐mediated end‐joining (MMEJ) . However, none of these studies assessed the effects of the characteristic sequences of MARs on transgene expression.…”

“…Scaffold/matrix attachment regions (S/MARs) can block transgene silencing [6][7][8] and increase transgene expression levels and stability in host cells [9][10][11][12][13][14][15]. In addition, S/MARs can also reduce variations in transgene expression among different cells to some extent, and the rate of transgene genomic integration can be increased [11].…”

Key Challenges in Designing CHO Chassis Platforms

“…Consequently, the resulting productivity may vary, as it was shown that adjacent chromatin states can spread into newly integrated sequences. To avoid this, genetic sequences that maintain an open chromatin configuration have been used [195,196]. Direct silencing of the CMV promoter used for most transgenes, either by DNA-methylation or by changes in the histone modifications has also been shown [197][198][199][200].…”