Context-dependent genomic locus effects on antibody production in recombinant Chinese hamster ovary cells generated through random integration.

High-yield production of therapeutic protein using Chinese hamster ovary (CHO) cells requires stable cell line development (CLD). CLD typically uses random integration of transgenes; however, this results in clonal variation and subsequent laborious clone screening. Therefore, site-specific integration of a protein expression cassette into a desired chromosomal locus showing high transcriptional activity and stability, referred to as a hot spot, is emerging. Although positional effects are important for therapeutic protein expression, the sequence-specific mechanisms by which hotspots work are not well understood. In this study, we performed whole-genome sequencing (WGS) to locate randomly inserted vectors in the genome of recombinant CHO cells expressing high levels of monoclonal antibodies (mAbs) and experimentally validated these locations and vector compositions. The integration site was characterized by active histone marks and potential enhancer activities, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) mediated indel mutations in the region upstream of the integration site led to a significant reduction in specific antibody productivity by up to 30%. Notably, the integration site and its core region did not function equivalently outside the native genomic context, showing a minimal effect on the increase in exogenous protein expression in the host cell line. We also observed a superior production capacity of the mAb expressing cell line compared to that of the host cell line. Collectively, this study demonstrates that developing recombinant CHO cell lines to produce therapeutic proteins at high levels requires a balance of factors including transgene configuration, genomic locus landscape, and host cell properties.

Knockout of the lysosomal membrane protein, LAMP2C, improves transient gene expression in HEK293 cells via increased intracellular plasmid availability.

Plasmid-based transfection can be used in many applications such as transient gene expression (TGE)-based therapeutic protein production. These applications preferentially require maximization of intracellular plasmid availability. Here, we applied a lysosome engineering approach to alleviate lysosome-mediated nucleic acid degradation and enhance the TGE in mammalian cells. By knocking out the lysosomal membrane protein LAMP2C, which is known to be the main player in RNautophagy/DNautophagy (RDA), we significantly improved transient fluorescent protein expression in HEK293 cells by improving the retention rate of transfected plasmids; however, this effect was not observed in CHO cells. Additional knockout of a lysosomal membrane transporter and another RDA player, SIDT2, was ineffective, regardless of the presence of LAMP2C. LAMP2C knockout enhanced TGE-based mAb production in HEK293 cells by up to 2.82-fold increase in specific mAb productivity. Taken together, these results demonstrate that HEK293 cells can be engineered to improve the usage of the transfected plasmid via knockout of the lysosomal membrane protein LAMP2C and provide efficient host cells in TGE systems for therapeutic protein production.

Implementation of ubiquitous chromatin opening elements as artificial integration sites for CRISPR/Cas9-mediated knock-in in mammalian cells.

CRISPR/Cas9-mediated targeted gene integration (TI) has been used to generate recombinant mammalian cell lines with predictable transgene expression. Identifying genomic hot spots that render high and stable transgene expression and knock-in (KI) efficiency is critical for fully implementing TI-mediated cell line development (CLD); however, such identification is cumbersome. In this study, we developed an artificial KI construct that can be used as a hot spot at different genomic loci. The ubiquitous chromatin opening element (UCOE) was employed because of its ability to open chromatin and enable stable and site-independent transgene expression. UCOE KI cassettes were randomly integrated into CHO-K1 and HEK293T cells, followed by TI of enhanced green fluorescent protein (EGFP) onto the artificial UCOE KI site. The CHO-K1 random pool harboring 5'2.2A2UCOE-CMV displayed a significant increase in EGFP expression level and KI efficiency compared with that of the control without UCOE. In addition, 5'2.2A2UCOE-CMV showed improved Cas9 accessibility in the HEK293T genome, leading to an increase in indel frequency and homology-independent KI. Overall, this assessment revealed the potential of UCOE KI constructs as artificial integration sites in streamlining the screening of high-production targeted integrants by mitigating the selection of genomic hot spots.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells.

Site-specific integration has emerged as a promising strategy for streamlined and predictable Chinese hamster ovary (CHO) cell line development (CLD). However, the low specific productivity of the targeted integrants limits their practical application. In this study, we developed a hybrid CLD platform combining site-specific integration of a transgene and dihydrofolate reductase/methotrexate (DHFR/MTX)-mediated gene amplification to generate high-producing recombinant CHO cell lines. We used the CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform to integrate the DHFR expression cassette and transgene landing pad into a CHO genomic hot spot, C12orf35 locus, of DHFR-knockout CHO-K1 host cell lines. When subjected to various MTX concentrations up to 1 µM, EGFP-expressing targeted integrants showed a 3.6-fold increase in EGFP expression in the presence of 200 nM MTX, accompanied by an increase in the DHFR and EGFP copy number. A single-step 200 nM MTX amplification increased the specific monoclonal antibody (mAb) productivity (q mAb ) of recombinant mAb-producing targeted integrants by 2.8-folds, reaching a q mAb of 9.1-11.0 pg/cell/day. Fluorescence in situ hybridization analysis showed colocalization of DHFR and mAb sequences at the intended chromosomal locations without clear amplified arrays of signals. Most MTX-amplified targeted integrants sustained recombinant mAb production during long-term culture in the absence of MTX, supporting stable gene expression in the amplified cell lines. Our study provides a new CLD platform that increases the productivity of targeted integrants by amplifying the transgene copies.

Platyconic Acid A, Platycodi Radix-Derived Saponin, Suppresses TGF-1-induced Activation of Hepatic Stellate Cells via Blocking SMAD and Activating the PPAR Signaling Pathway.

Platycodi radix is a widely sold health food worldwide, which contains numerous phytochemicals that are beneficial to health. Previously, we reported that saponin from the roots of Platycodi radix-derived saponin inhibited toxicant-induced liver diseases. Nevertheless, the inhibitory effect of platyconic acid A (PA), the active component of Platycodi radix-derived saponin, on the anti-fibrotic activity involving the SMAD pathway remains unclear. We investigated the inhibitory effects of PA on TGF-1-induced activation of hepatic stellate cells (HSCs). PA inhibited TGF-1-enhanced cell proliferation, as well as expression of -SMA and collagen 1 in HSC-T6 cells. PA suppressed TGF-1-induced smad2/3 phosphorylation and smad binding elements 4 (SBE4) luciferase activity. Reversely, PA restored TGF-1-reduced expression of smad7 and peroxisome proliferator-activated receptor (PPAR)γ. PA also repressed TGF-1-induced phosphorylation of Akt and MAPKs. In summary, the results suggest that the inhibitory effect of PA on HSCs occurs through the blocking of SMAD-dependent and SMAD-independent pathways, leading to the suppression of -SMA and collagen 1 expression.