Enhancing the production of adeno-associated virus (AAV)2 and AAV9 with high full capsid ratio in HEK293 cells through design-of-experiment optimization of triple plasmid ratio.

The recombinant adeno-associated virus (rAAV) vectors used in gene therapy are usually produced by transfecting three different plasmids (Adenoviral helper plasmid (pHelper), AAV rep/cap plasmids (pRepCap), and Transgene plasmid (pAAV-GOI)) into human embryonic kidney 293 (HEK293) cells. However, the high proportion of unwanted empty capsids generated during rAAV production is problematic. To simultaneously enhance the genome titer and full capsid ratio, the ratio of the three plasmids transfected into HEK293 cells was optimized using design-of-experiment (DoE). AAV2 and AAV9, which have different production kinetics, were selected as cell-associated and secreted model AAVs, respectively. In 125 mL Erlenmeyer flasks, the genome titers of rAAV2 and rAAV9 at DoE-optimized plasmid weight ratios (pHelper:pRep2Cap2:pAAV-GOI = 1:3.52:0.50 for rAAV2 and pHelper:pRep2Cap9:pAAV-GOI = 1:1.44:0.27 for rAAV9) were 2.23-fold and 2.26-fold higher than those in the widely used plasmid weight ratio (1:1:1), respectively. In addition, compared with the plasmid ratio of 1:1:1, the relative VP3 band intensities of rAAV2 and rAAV9, which represent the relative empty capsid ratios, were reduced by 26% and 25%, respectively, at the DoE-optimized plasmid ratio. Reduced empty capsid ratios in the DoE-optimized plasmid ratios were also confirmed using transmission electron microscopy (TEM). Taken together, regardless of the AAV serotype, DoE-aided optimization of the triple plasmid ratio was found to be an efficient means of improving the production of rAAV with a high full capsid ratio.

Effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoprotein in recombinant CHO cells.

The occurrence of autophagy in recombinant Chinese hamster ovary (rCHO) cell culture has attracted attention due to its effects on therapeutic protein production. Given the significance of glycosylation in therapeutic proteins, this study examined the effects of autophagy-inhibiting chemicals on sialylation of Fc-fusion glycoproteins in rCHO cells. Three chemical autophagy inhibitors known to inhibit different stages were separately treated with two rCHO cell lines that produce the same Fc-fusion glycoprotein derived from DUKX-B11 and DG44. All autophagy inhibitors significantly decreased the sialylation of Fc-fusion glycoprotein in both cell lines. The decrease in sialylation of Fc-fusion glycoprotein is unlikely to be attributed to the release of intracellular enzymes, given the high cell viability and low activity of extracellular sialidases. Interestingly, the five intracellular nucleotide sugars remained abundant in cells treated with autophagy inhibitors. In the mRNA expression profiles of 27 N-glycosylation-related genes using the NanoString nCounter system, no significant differences in gene expression were noted. With the positive effect of supplementing nucleotide sugar precursors on sialylation, attempts were made to enhance the levels of intracellular nucleotide sugars by supplying these precursors. The addition of nucleotide sugar precursors to cultures treated with inhibitors successfully enhanced the sialylation of Fc-fusion glycoproteins compared to the control culture. This was particularly evident under mild stress conditions and not under relatively severe stress conditions, which were characterized by a high decrease in sialylation. These results suggest that inhibiting autophagy in rCHO cell culture decreases sialylation of Fc-fusion glycoprotein by constraining the availability of intracellular nucleotide sugars. KEY POINTS: •  The autophagy inhibition in rCHO cell culture leads to a significant reduction in the sialylation of Fc-fusion glycoprotein. •  The pool of five intracellular nucleotide sugars remained highly abundant in cells treated with autophagy inhibitors. •  Supplementation of nucleotide sugar precursors effectively restores decreased sialylation, particularly under mild stress conditions but not in relatively severe stress conditions.

Genome-Wide CRISPR/Cas9 Screening Unveils a Novel Target ATF7IP-SETDB1 Complex for Enhancing Difficult-to-Express Protein Production.

With the emerging novel biotherapeutics that are typically difficult-to-express (DTE), improvement is required for high-yield production. To identify novel targets that can enhance DTE protein production, we performed genome-wide fluorescence-activated cell sorting (FACS)-based clustered regularly interspaced short palindromic repeats (CRISPR) knockout screening in bispecific antibody (bsAb)-producing Chinese hamster ovary (CHO) cells. The screen identified the two highest-scoring genes, Atf7ip and Setdb1, which are the binding partners for H3K9me3-mediated transcriptional repression. The ATF7IP-SETDB1 complex knockout in bsAb-producing CHO cells suppressed cell growth but enhanced productivity by up to 2.7-fold. Decreased H3K9me3 levels and an increased transcriptional expression level of the transgene were also observed. Furthermore, perturbation of the ATF7IP-SETDB1 complex in monoclonal antibody (mAb)-producing CHO cells led to substantial improvements in mAb production, increasing the productivity by up to 3.9-fold without affecting the product quality. Taken together, the genome-wide FACS-based CRISPR screen identified promising targets associated with histone methylation, whose perturbation enhanced the productivity by unlocking the transgene expression.

Genome-wide CRISPR/Cas9 knockout screening to mitigate cell growth inhibition induced by histone deacetylase inhibitors in recombinant CHO cells.

Histone deacetylase inhibitors (iHDACs) have been extensively studied as enhancers of therapeutic protein production in recombinant Chinese hamster ovary (CHO) (rCHO) cell cultures. However, the addition of iHDACs reduces the viable cell concentration (VCC) in rCHO cell cultures, thereby reducing their potential to enhance therapeutic protein production. To mitigate the negative effects of iHDACs on VCC, screening using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-based single-gene knockout (KO) library in rCHO cells was performed in the presence of CI994, a member of iHDACs, and 10 potential KO genes that enhanced the VCC of CI994-treated rCHO cells were identified. Among these, Bcor was validated as a promising KO target that improved VCC without negatively affecting the specific productivity in the presence of CI994. Bcor KO increased the VCC and therapeutic protein concentrations in both batch and fed-batch cultures in the presence of CI994. Taken together, these findings highlight the potential of the whole-genome CRISPR/Cas9-based single-gene KO cell library to identify KO target genes for the development of iHDAC-resistant rCHO cells for enhanced therapeutic protein production.

Enhancing the production of recombinant human TGF-ß1 through an understanding of TGF-ß1 synthesis, signaling, and endocytosis in CHO cells.

To enhance the production of recombinant human transforming growth factor-beta1 (rhTGF-ß1) in Chinese hamster ovary (CHO) cells, rhTGF-ß1 was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature rhTGF-ß1 used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature rhTGF-ß1 was mostly produced in the form of latent rhTGF-ß1 in cultures of recombinant CHO (rCHO) cells producing rhTGF-ß1 (CHO-rhTGF-ß1). The concentration of active mature rhTGF-ß1 in the culture supernatant of CHO-rhTGF-ß1 cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by CHO-rhTGF-ß1 cells. Overexpression of PACEsol, a soluble form of furin, in CHO-rhTGF-ß1 cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature rhTGF-ß1 concentration (6.4 µg mL-1 ) was obtained with the PACEsol-expressing clone, which was approximately 45% higher than that of the parental clone (P < 0.01). Thus, a comprehensive understanding of the intrinsic properties of rhTGF-ß1 with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature rhTGF-ß1 in CHO cells.

In vitro induction of in vivo-relevant stellate astrocytes in 3D brain-derived, decellularized extracellular matrices.

In vitro fabrication of 3D cell culture systems that could provide in vivo tissue-like, structural, and biochemical environments to neural cells is essential not only for fundamental studies on brain function and behavior, but also for tissue engineering and regenerative medicine applicable to neural injury and neurodegenerative diseases. In particular, for astrocytes-which actively respond to the surroundings and exhibit varied morphologies based on stimuli (e.g., stiffness and chemicals) in vitro, as well as physiological or pathological conditions in vivo-it is crucial to establish an appropriate milieu in in vitro culture platforms. Herein, we report the induction of in vivo-relevant, stellate-shaped astrocytes derived from cortices of Rattus norvegicus by constructing the 3D cell culture systems of brain-derived, decellularized extracellular matrices (bdECMs). The bdECM hydrogels were mechanically stable and soft, and the bdECM-based 3D scaffolds supplied biochemically active environments that astrocytes could interact with, leading to the development of in vivo-like stellate structures. In addition to the distinct morphology with actively elongated endfeet, the astrocytes, cultured in 3D bdECM scaffolds, would have neurosupportive characteristics, indicated by the accelerated neurite outgrowth in the astrocyte-conditioned media. Furthermore, next-generation sequencing showed that the gene expression profiles of astrocytes cultured in bdECMs were significantly different from those cultured on 2D surfaces. The stellate-shaped astrocytes in the bdECMs were analyzed to have reached a more mature state, for instance, with decreased expression of genes for scaffold ECMs, actin filaments, and cell division. The results suggest that the bdECM-based 3D culture system offers an advanced platform for culturing primary cortical astrocytes and their mixtures with other neural cells, providing a brain-like, structural and biochemical milieu that promotes the maturity and in vivo-like characteristics of astrocytes in both form and gene expression. STATEMENT OF SIGNIFICANCE: Decellularized extracellular matrices (dECMs) have emerged as strong candidates for the construction of three-dimensional (3D) cell cultures in vitro, owing to the potential to provide native biochemical and physical environments. In this study, we fabricated hydrogels of brain-derived dECMs (bdECMs) and cultured primary astrocytes within the bdECM hydrogels in a 3D context. The cultured astrocytes exhibited a stellate morphology distinct from conventional 2D cultures, featuring tridimensionally elongated endfeet. qRT-PCR and NGS-based transcriptomic analyses revealed gene expression patterns indicative of a more mature state, compared with the 2D culture. Moreover, astrocytes cultured in bdECMs showed neurosupportive characteristics, as demonstrated by the accelerated neurite outgrowth in astrocyte-conditioned media. We believe that the bdECM hydrogel-based culture system can serve as an in vitro model system for astrocytes and their coculture with other neural cells, holding significant potential for neural engineering and therapeutic applications.

Identification of hyperosmotic stress-responsive genes in Chinese hamster ovary cells via genome-wide virus-free CRISPR/Cas9 screening.

Chinese hamster ovary (CHO) cells are the preferred mammalian host cells for therapeutic protein production that have been extensively engineered to possess the desired attributes for high-yield protein production. However, empirical approaches for identifying novel engineering targets are laborious and time-consuming. Here, we established a genome-wide CRISPR/Cas9 screening platform for CHO-K1 cells with 111,651 guide RNAs (gRNAs) targeting 21,585 genes using a virus-free recombinase-mediated cassette exchange-based gRNA integration method. Using this platform, we performed a positive selection screening under hyperosmotic stress conditions and identified 180 genes whose perturbations conferred resistance to hyperosmotic stress in CHO cells. Functional enrichment analysis identified hyperosmotic stress responsive gene clusters, such as tRNA wobble uridine modification and signaling pathways associated with cell cycle arrest. Furthermore, we validated 32 top-scoring candidates and observed a high rate of hit confirmation, demonstrating the potential of the screening platform. Knockout of the novel target genes, Zfr and Pnp, in monoclonal antibody (mAb)-producing recombinant CHO (rCHO) cells and bispecific antibody (bsAb)-producing rCHO cells enhanced their resistance to hyperosmotic stress, thereby improving mAb and bsAb production. Overall, the collective findings demonstrate the value of the screening platform as a powerful tool to investigate the functions of genes associated with hyperosmotic stress and to discover novel targets for rational cell engineering on a genome-wide scale in CHO cells.

Mitigating transcriptional bottleneck using a constitutively active transcription factor, VP16-CREB, in mammalian cells.

High-level expression of recombinant proteins in mammalian cells has long been an area of interest. Inefficient transcription machinery is often an obstacle in achieving high-level expression of recombinant proteins in mammalian cells. Synthetic promoters have been developed to improve the transcription efficiency, but have achieved limited success due to the limited availability of transcription factors (TFs). Here, we present a TF-engineering approach to mitigate the transcriptional bottlenecks of recombinant proteins. This includes: (i) identification of cAMP response element binding protein (CREB) as a candidate TF by searching for TFs enriched in the cytomegalovirus (CMV) promoter-driven high-producing recombinant Chinese hamster ovary (rCHO) cell lines via transcriptome analysis, (ii) confirmation of transcriptional limitation of active CREB in rCHO cell lines, and (iii) direct activation of the transgene promoter by expressing constitutively active CREB at non-cytotoxic levels in rCHO cell lines. With the expression of constitutively active VP16-CREB, the production of therapeutic proteins, such as monoclonal antibody and etanercept, in CMV promoter-driven rCHO cell lines was increased up to 3.9-fold. VP16-CREB was also used successfully with synthetic promoters containing cAMP response elements. Taken together, this strategy to introduce constitutively active TFs into cells is a useful means of overcoming the transcriptional limitations in recombinant mammalian cells.

Streamlined in vitro screening system of synthetic signal peptides in Chinese hamster ovary cells for therapeutic protein production.

Increasing the screening efficiency and maintaining the N-terminal cleavage pattern are key factors in the development of an in vitro synthetic signal peptide screening system for high therapeutic protein production in Chinese hamster ovary (CHO) cells. This study improved the in vitro screening system of synthetic signal peptides in CHO cells for therapeutic protein production by modifying the expression vector. Incorporating a leaky stop codon with IgG transmembrane and cytoplasmic domains into the expression vector improved the proportion of high producers in establishing stable CHO cell pools. The selected signal peptides from stable CHO cell pools that were generated using degenerate codon-based oligonucleotides with a conserved polar carboxy-terminal domain in the native signal peptide showed similar N-terminal cleavage patterns to the native one. In addition, replacing native signal peptide with selected synthetic signal peptides did not influence the sialylated N-linked glycan formation and biological activity of therapeutic Fc-fusion glycoprotein in CHO cells. Thus, an in vitro synthetic signal peptide screening system can be used for therapeutic Fc-fusion glycoprotein production in CHO cells with an enhanced specific protein productivity while maintaining the N-terminal cleavage pattern similar to the native one.

Enhanced cell growth, production, and mAb quality produced in Chinese hamster ovary-K1 cells by supplementing polyamine in the media.

Polyamines such as putrescine (PUT), spermidine (SPD), and spermine (SPM) are amine group-containing biomolecules that regulate multiple intracellular functions such as proliferation, differentiation, and stress response in mammalian cells. Although these biomolecules can be generated intracellularly, lack of polyamine-synthesizing activity has occasionally been reported in a few mammalian cell lines such as Chinese hamster ovary (CHO)-K1; thus, polyamine supplementation in serum-free media is required to support cell growth and production. In the present study, the effects of biogenic polyamines PUT, SPD, and SPM in media on cell growth, production, metabolism, and antibody quality were explored in cultures of antibody-producing CHO-K1 cells. Polyamine withdrawal from media significantly suppressed cell growth and production. On the other hand, enhanced culture performance was achieved in polyamine-containing media conditions in a dose-dependent manner regardless of polyamine type. In addition, in polyamine-deprived medium, distinguishing metabolic features, such as enriched glycolysis and suppressed amino acid consumption, were observed and accompanied by higher heterogeneity of antibody quality compared with the optimal concentration of polyamines. Furthermore, an excessive concentration of polyamines negatively affected culture performance as well as antibody quality. Hence, the results suggest that polyamine-related metabolism needs to be further investigated and polyamines in cell growth media should be optimized as a controllable parameter in CHO cell culture bioprocessing. KEY POINTS: • Polyamine supplementation enhanced cell growth and production in a dose-dependent manner • Polyamine type and concentration in the media affected mAb quality • Optimizing polyamines in the media is suggested in CHO cell bioprocessing.

Improving bone morphogenetic protein (BMP) production in CHO cells through understanding of BMP synthesis, signaling and endocytosis.

Bone morphogenetic proteins (BMPs) are a group of growth factors with the clinical potential to regulate cartilage and bone formation. Functionally active mature recombinant human BMPs (rhBMPs), produced primarily in Chinese hamster ovary (CHO) cells for clinical applications, are considered difficult to express because they undergo maturation processes, signaling pathways, or endocytosis. Although BMPs are a family of proteins with similar mature domain sequence identities, their individual properties are diverse. Thus, understanding the properties of individual rhBMPs is essential to improve rhBMP production in CHO cells. In this review, we discuss various approaches to improve rhBMP production in CHO cells by understanding the overall maturation process, signaling pathways and endocytosis of individual rhBMPs.

Enhancing CHO cell productivity through a dual selection system using Aspg and Gs in glutamine free medium.

The dominant method for generating Chinese hamster ovary (CHO) cell lines that produce high titers of biotherapeutic proteins utilizes selectable markers such as dihydrofolate reductase (Dhfr) or glutamine synthetase (Gs), alongside inhibitory compounds like methotrexate or methionine sulfoximine, respectively. Recent work has shown the importance of asparaginase (Aspg) for growth in media lacking glutamine-the selection medium for Gs-based selection systems. We generated a Gs/Aspg double knockout CHO cell line and evaluated its utility as a novel dual selectable system via co-transfection of Gs-Enbrel and Aspg-Enbrel plasmids. Using the same selection conditions as the standard Gs system, the resulting cells from the Gs/Aspg dual selection showed substantially improved specific productivity and titer compared to the standard Gs selection method, however, with reduced growth rate and viability. Following adaptation in the selection medium, the cells improved viability and growth while still achieving ~5-fold higher specific productivity and ~3-fold higher titer than Gs selection alone. We anticipate that with further optimization of culture medium and selection conditions, this approach would serve as an effective addition to workflows for the industrial production of recombinant biotherapeutics.

Development of an in vitro screening system for synthetic signal peptide in mammalian cell-based protein production.

Optimizing appropriate signal peptides in mammalian cell-based protein production is crucial given that most recombinant proteins produced in mammalian cells are thought to be secreted proteins. Until now, most studies on signal peptide in mammalian cells have replaced native signal peptides with well-known heterologous signal peptides and bioinformatics-based signal peptides. In the present study, we successfully established an in vitro screening system for synthetic signal peptide in CHO cells by combining a degenerate codon-based oligonucleotides library, a site-specific integration system, and a FACS-based antibody detection assay. Three new signal peptides were screened using this new screening system, confirming to have structural properties as signal peptides by the SignalP web server, a neural network-based algorithm that quantifies the signal peptide-ness of amino acid sequences. The novel signal peptides selected in this study increased Fc-fusion protein production in CHO cells by increasing specific protein productivity, whereas they did not negatively affect cell growth. Particularly, the SP-#149 clone showed the highest qp, 0.73 ± 0.01 pg/cell/day from day 1 to day 4, representing a 1.47-fold increase over the native signal peptide in a serum-free suspension culture mode. In addition, replacing native signal peptide with the novel signal peptides did not significantly affect sialylated N-glycan formation, N-terminal cleavage pattern, and biological function of Fc-fusion protein produced in CHO cells. The overall results indicate the utility of a novel in vitro screening system for synthetic signal peptide for mammalian cell-based protein production. KEY POINTS: • An in vitro screening system for synthetic signal peptide in mammalian cells was established • This system combined a degenerate codon-based library, site-specific integration, and a FACS-based detection assay • The novel signal peptides selected in this study could increase Fc-fusion protein production in mammalian cells.

Recombinase-mediated cassette exchange-based screening of a CRISPR/Cas9 library for enhanced recombinant protein production in human embryonic kidney cells: Improving resistance to hyperosmotic stress.

Targeted engineering of mammalian cells has been widely attempted to ensure the efficient production of therapeutic proteins with proper quality during bioprocesses. However, the identification of novel targets for cell engineering is labor-intensive and has not yet been fully substantiated. Here, we established a CRISPR/Cas9 library screening platform in human embryonic kidney (HEK293) cells based on guide RNA integration mediated by recombinase-mediated cassette exchange (RMCE) to interrogate gene function in a high-throughput manner. This platform was further advanced using a nuclear localization signal-tagged recombinase that increased RMCE efficiency by 4.8-fold. Using this platform, we identified putative target genes, such as CDK8, GAS2L1, and GSPT1, and their perturbation confers resistance to hyperosmotic stress that inhibits cell growth and induces apoptosis. Knockout of these genes in monoclonal antibody (mAb)-producing recombinant HEK293 (rHEK293) cells enhanced resistance to hyperosmotic stress-induced apoptosis, resulting in enhanced mAb production. In particular, GSPT1-knockout yielded 2.3-fold increase in maximum mAb concentration in fed-batch culture where hyperosmotic stress naturally occurs due to nutrient feeding. Taken together, this streamlined screening platform allows the identification of novel targets associated with hyperosmotic stress, enabling the development of stress-resistant cells producing recombinant proteins.

Factors affecting the quality of therapeutic proteins in recombinant Chinese hamster ovary cell culture.

Chinese hamster ovary (CHO) cells are the most widely used mammalian host cells for the commercial production of therapeutic proteins. Fed-batch culture is widely used to produce therapeutic proteins, including monoclonal antibodies, because of its operational simplicity and high product titer. Despite technical advances in the development of culture media and cell cultures, it is still challenging to maintain high productivity in fed-batch cultures while also ensuring good product quality. In this review, factors that affect the quality attributes of therapeutic proteins in recombinant CHO (rCHO) cell culture, such as glycosylation, charge variation, aggregation, and degradation, are summarized and categorized into three groups: culture environments, chemical additives, and host cell proteins accumulated in culture supernatants. Understanding the factors that influence the therapeutic protein quality in rCHO cell culture will facilitate the development of large-scale, high-yield fed-batch culture processes for the production of high-quality therapeutic proteins.

Improving the secretory capacity of CHO producer cells: The effect of controlled Blimp1 expression, a master transcription factor for plasma cells.

With the advent of novel therapeutic proteins with complex structures, cellular bottlenecks in secretory pathways have hampered the high-yield production of difficult-to-express (DTE) proteins in CHO cells. To mitigate their limited secretory capacity, recombinant CHO (rCHO) cells were engineered to express Blimp1, a master regulator orchestrating B cell differentiation into professional secretory plasma cells, using the streamlined CRISPR/Cas9-based recombinase-mediated cassette exchange landing pad platform. The expression of Blimp1α or Blimp1ß in rCHO cells producing DTE recombinant human bone morphogenetic protein-4 (rhBMP-4) increased specific rhBMP-4 productivity (qrhBMP-4). However, since Blimp1α expression suppressed cell growth more significantly than Blimp1ß expression, only Blimp1ß expression enhanced rhBMP-4 yield. In serum-free suspension culture, Blimp1ß expression significantly increased the rhBMP-4 concentration (>3-fold) and qrhBMP-4 (>4-fold) without significant increase in hBMP-4 transcript levels. In addition, Blimp1ß expression facilitated mature rhBMP-4 secretion by active proteolytic cleavage in the secretory pathway. Transcriptomic profiling (RNA-seq) revealed global changes in gene expression patterns that promote protein processing in secretory organelles. In-depth integrative analysis of the current RNA-seq data, public epigenome/RNA-seq data, and in silico analysis identified 45 potential key regulators of Blimp1 that are consistently up- or down-regulated in Blimp1ß expressing rCHO cells and plasma cells. Blimp1ß expression also enhanced the production of easy-to-express monoclonal antibodies (mAbs) and modulated the expression of key regulators in rCHO cells producing mAb. Taken together, the results show that controlled expression of Blimp1ß improves the production capacity of rCHO cells by regulating secretory machinery and suggest new opportunities for engineering promising targets that are resting in CHO cells.

Small molecule epigenetic modulators for enhancing recombinant antibody production in CHO cell cultures.

Small molecule epigenetic modulators that modify epigenetic states in cells are useful tools for regulating gene expression by inducing chromatin remodeling. To identify small molecule epigenetic modulators that enhance recombinant protein expression in Chinese hamster ovary (CHO) cells, we examined eight histone deacetylase inhibitors (iHDACs) and six DNA methyltransferase inhibitors as chemical additives in recombinant CHO (rCHO) cell cultures. Among these, a benzamide-based iHDAC, CI994, was the most effective in increasing monoclonal antibody (mAb) production. Despite suppressing cell growth, the addition of CI994 to mAb-expressing GSR cell cultures at 10 µM resulted in a 2.3-fold increase in maximum mAb concentration due to a 3.0-fold increase in specific mAb productivity (qmAb ). CI994 increased mAb messenger RNA levels and histone H3 acetylation in GSR cells, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that CI994 significantly increased the histone H3 acetylation level at the cytomegalovirus promoter driving mAb gene expression, indicating that chromatin remodeling in the promoter region results in enhanced mAb gene transcription and qmAb . Similar beneficial effects of CI994 on mAb production were observed in mAb-expressing CS13-1.00 cells. Collectively, our findings indicate that CI994 increases mAb production in rCHO cell cultures by chromatin remodeling resulting from acetylation of histones in the mAb gene promoter.

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

Pooled CRISPR screens have been widely applied to mammalian and other organisms to elucidate the interplay between genes and phenotypes of interest. The most popular method for delivering the CRISPR components into mammalian cells is lentivirus based. However, because lentivirus is not always an option, virus-free protocols are starting to emerge. Here, we demonstrate an improved virus-free, genome-wide CRISPR screening platform for Chinese hamster ovary cells with 75,488 gRNAs targeting 15,028 genes. Each gRNA expression cassette in the library is precisely integrated into a genomic landing pad, resulting in a very high percentage of single gRNA insertions and minimal clonal variation. Using this platform, we perform a negative selection screen on cell proliferation that identifies 1,980 genes that affect proliferation and a positive selection screen on the toxic endoplasmic reticulum stress inducer, tunicamycin, that identifies 77 gene knockouts that improve survivability.

Blockage of undesirable endocytosis of recombinant human growth/differentiation factor-5 in Chinese hamster ovary cell cultures requires heparin analogs with specific chain lengths.

Cell surface heparan sulfate proteoglycan (HSPG)-mediated endocytosis lowers the yield of recombinant human bone morphogenetic proteins (rhBMPs), such as rhBMP-2 and rhBMP-4, from Chinese hamster ovary (CHO) cell cultures. Exogenous recombinant human growth/differentiation factor-5 (rhGDF-5), a member of the BMP family, bound to cell surface HSPGs and was actively internalized into CHO cells. Knockdown of heparan sulfate (HS) synthesis enzymes in CHO cells revealed that the chain length and N-sulfation of HS affected the binding of rhGDF-5 to HSPGs and subsequent rhGDF-5 internalization. To increase product yield by minimizing rhGDF-5 internalization in recombinant CHO (rCHO) cell cultures, heparin, and dextran sulfate (DS) of various polysaccharide chain lengths, which are structural analogs of HS, were examined for blockage of rhGDF-5 internalization. Heparin fragments of four monosaccharides (MW of 1.2 kDa) and DS (MW of 15 kDa) did not inhibit rhGDF-5 internalization whereas unfractionated heparin and DS of 200 kDa could significantly inhibit it. Compared to the control cultures, supplementation with unfractionated heparin or DS of 200 kDa at 1 g L-1 resulted in more than a 10-fold increase in the maximum rhGDF-5 concentration. Taken together, the supplementation of structural HS analogs improved rhGDF-5 production in rCHO cell cultures by inhibiting rhGDF-5 internalization.

A Chinese hamster transcription start site atlas that enables targeted editing of CHO cells.

Chinese hamster ovary (CHO) cells are widely used for producing biopharmaceuticals, and engineering gene expression in CHO is key to improving drug quality and affordability. However, engineering gene expression or activating silent genes requires accurate annotation of the underlying regulatory elements and transcription start sites (TSSs). Unfortunately, most TSSs in the published Chinese hamster genome sequence were computationally predicted and are frequently inaccurate. Here, we use nascent transcription start site sequencing methods to revise TSS annotations for 15 308 Chinese hamster genes and 3034 non-coding RNAs based on experimental data from CHO-K1 cells and 10 hamster tissues. We further capture tens of thousands of putative transcribed enhancer regions with this method. Our revised TSSs improves upon the RefSeq annotation by revealing core sequence features of gene regulation such as the TATA box and the Initiator and, as exemplified by targeting the glycosyltransferase gene Mgat3, facilitate activating silent genes by CRISPRa. Together, we envision our revised annotation and data will provide a rich resource for the CHO community, improve genome engineering efforts and aid comparative and evolutionary studies.