A novel Bxb1 integrase RMCE system for high fidelity site‐specific integration of mAb expression cassette in CHO Cells

Abstract

As CHO cell line development for biotherapeutic production becomes more sophisticated through the availability of the CHO genome sequence, the ability to accurately and reproducibly engineer the host cell genome has become increasingly important. Multiple well characterized systems for site-specific integration will enable more complex cell line engineering to generate cell lines with desirable attributes. We built and characterized a novel recombinase mediated cassette exchange (RMCE) system using Bxb1 integrase and compared it to the commonly used Flp/FRT RMCE system. We first integrated a DNA construct flanked by either Bxb1 attachment sites or FRT sequences (referred to as a landing pad) into the Fer1L4 genomic locus of CHO-S cells using CRISPR/Cas9 mediated homologous recombination. We characterized the resulting clones harboring either the Bxb1 or Flp/FRT landing pad using whole genome resequencing to compare their genomes with the parental host cell line. We determined that each landing pad was specifically integrated into the Fer1L4 locus in the selected clones and observed no major structural changes in the genome or variations in copy number as a result of CRISPR/Cas9 modification. We subsequently tested the ability of the Bxb1 and Flp/FRT landing pad clones to perform proper RMCE with donor vectors containing identical mAb expression cassettes flanked by either Bxb1 attachment sites or FRT sites. We demonstrated that both RMCE systems were able to generate stable pools in a similar time frame with comparable mAb expression. Through genetic characterization of up to 24 clones derived from either system, we determined that the BxB1 RMCE system yielded higher fidelity RMCE events than the Flp/FRT system as evidenced by a higher percentage of clones with expected integration of the mAb cassette into the landing pad in the respective cell lines. We conclude that Bxb1 RMCE is an excellent alternative to Flp/FRT RMCE and valuable addition to our toolbox enabling the engineering of more sophisticated cell lines for biotherapeutic production. Biotechnol. Bioeng. 2017;114: 1837-1846. © 2017 Wiley Periodicals, Inc.

Single Copy Transgene Integration in a Transcriptionally Active Site for Recombinant Protein Synthesis

“…This may contribute to the variability. Other studies using RMCE to insert a product gene into a defined landing pad have also seen variations among producing clones . Few had quantified the transgene transcript level, and each had utilized different products and integration sites.…”

“…A number of methods have been developed for RMCE using site specific recombinases, such as the Cre/ lox, Flp/ FRT, and ϕC31/ att P/‐B systems . Integration of transgenes into CHO cells has been achieved at a reasonable efficiency using both targeted integration and cassette exchange . Another similar method, dual RMCE, utilizes two different recombinases (such as Flp and Cre).…”

Recurring genomic structural variation leads to clonal instability and loss of productivity

“…With the advances in genome engineering, transgene integration can now be targeted to a specific locus in the host cell genome, without resorting to traditional selective pressure‐based transgene amplification (Cristea et al, ; Inniss et al, ; Lee, Grav, Pedersen, Lee, & Kildegaard, ; Lee, Kallehauge, Pedersen, & Kildegaard, ). In general, the site selected for targeted integration should be stable and not prone to structural variation or copy number changes.…”

Bottom-up approaches in synthetic biology and biomaterials for tissue engineering applications

“…Altogether, the ability to specifically target the cell genome using genome targeting genetic tools enable the placement of landing pads for the direct insertion of larger genetic circuits into the genome [174, 175]. These important advancements make it possible to program stem cells with genetic circuits capable of tightly regulating gene expression for directing their differentiation.…”