Host Cell Line Research Articles

The dual actions of miRNA16a in restricting Bovine Coronavirus replication through downregulation of Furin and enhancing the host immune response.

The roles of host cell miRNAs have not been well studied in the context of BCoV replication and immune regulation. This study aimed to identify miRNA candidates that regulate essential host genes involved in BCoV replication, tissue tropism, and immune regulation. To achieve these goals, we used two isolates of BCoV (enteric and respiratory) to infect bovine endothelial cells (BECs) and Madine Darby Bovine Kidney (MDBK) cells. We determined the miRNA expression profiles of these cells after BCoV infection. The expression of miRNA16a is differentially altered during BCoV infection. Our data show that miRNA16a is a significantly downregulated miRNA in both in vitro and ex vivo models. We confirmed the miRNA16aexpression profile by qRT-PCR. Overexpression of pre-miRNA16ain the BEC and the MDBK cell lines markedly inhibited BCoV infection, as determined by the viral genome copy numbers measured by qRT‒PCR, viral protein expression (S and N) measured by Western blot, and virus infectivity using a plaque assay. Our bioinformatic prediction showed that Furin is a potential target of miRNA16a. We compared the Furin protein expression level in pre-miRNA16a-transfected/BCoV-infected cells to that in pre-miRNA-scrambled-transfected cells. Our qRT-PCR and Western blot data revealed marked inhibition of Furin expression at the mRNA and protein levels, respectively. BCoV-S protein expression was markedly inhibited at both the mRNA and protein levels. To further confirm the impact of the downregulation of the Furin enzyme on the replication of BCoV, we transfected cells with specific Furin-siRNAs parallel to the scrambled siRNA. Marked inhibition of BCoV replication was observed in the Furin-siRNA-treated group. To further validate Furin as a novel target for miRNA16a, we cloned the 3'UTR of bovine Furin carrying the seed region of miRNA16a in the dual luciferase vector. Our data showed that luciferase activity in pre-miRNA16a-transfected cells decreased by more than 50% compared to cells transfected with the construct carrying the mutated Furin seed region. Our data confirmed that miRNA16ainhibits BCoV replication by targeting the host cell line Furin and the BCoV-S glycoprotein. It also enhances the host immune response, which contributes to the inhibition of viral replication. This is the first study to confirm that Furin is a valid target of miRNA16a. Our findings highlight the clinical applications of host miRNA16a as a potential miRNA-based vaccine/antiviral therapy.

Enhancing monoclonal antibody production efficiency using CHO-MK cells and specific media in a conventional fed-batch culture.

Chinese hamster ovary (CHO) cell lines, derived as subclones from the original CHO cell line, are widely used hosts for current biopharmaceutical productions. Recently, a highly proliferative host cell line, CHO-MK, was established from the Chinese hamster ovary tissue. In this study, we assessed the fundamental culture characteristics and capabilities of CHO-MK cells for monoclonal antibody (mAb) production using specified chemically defined media. To achieve this, we established fed-batch cultures of model CHO-MK cells in shake flasks and ambr15 and 2 L bioreactors under various conditions. The mAb-producing CHO-MK cell line A produced 12.6g/L of antibody within 7days in the fed-batch culture using a 2 L bioreactor, with a seeding density of 1 × 106 cells/mL. This performance corresponded to a space-time yield of 1.80g/L/day, representing a productivity level that could be challengingly attained in fed-batch cultures using conventional CHO cells. In addition, when we subjected six different mAb-producing CHO-MK cell lines to fed-batch culture in the ambr15 bioreactor for 7days, the antibody production ranged between 5.1 and 10.8g/L, confirming that combining CHO-MK cells and specified media leads to enhanced versatility. These discoveries underscore that CHO-MK cells combined with specified media might represent a next-generation production platform, which could potentially respond to an increasing demand for antibody drugs, reducing production costs, and shortening antibody drug development times. This study is expected to serve as a benchmark for future production process development using CHO-MK cells.

Characterization of the Ubiquitin-Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress.

Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER-stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC-MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO-DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N-glycan biosynthesis, and ubiquitin-mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER-stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

Characterizing the interplay between Acinetobacter baumannii, A549 cells, and anti-Omp34 antibodies: implications for adherence, internalization, and cytotoxicity.

Acinetobacter baumannii thrives within eukaryotic cells, influencing persistence, treatment approaches, and progression of disease. We probed epithelial cell invasion by A. baumannii and the influence of antibodies raised to outer membrane protein 34 (Omp34) on epithelial interactions. We expressed and purified recombinant Omp34 and induced anti-Omp34 antibodies in Bagg albino or BALB/c mice. Omp34 was evaluated for acute toxicity in mice through histological analysis of six organs. The host cell line, A549, was exposed to both A. baumannii 19606 and a clinical isolate. The study also investigated serum resistance, adherence, internalization, and proliferation of A. baumannii in A549 cells, with and without anti-Omp34 sera, utilizing cell culture techniques and light microscopy. A549 cell viability was evaluated by A. baumannii challenge and exposure to anti-Omp34 sera. Actin disruption experiments using cytochalasin D probed microfilament and microtubule roles in A. baumannii invasion. Omp34 prompted antibody production without toxicity in mice. The serum showed bactericidal effects on both strains. Additionally, both A. baumannii strains were found to form biofilms. Omp34 serum was observed to decrease biofilm formation, bacterial adherence, internalization, and proliferation in A549 cells. Furthermore, the use of anti-Omp34 serum enhanced the post-infection survival of the host cell. Pre-exposure of A549 cells to cytochalasin D reduced bacterial internalization, highlighting the role of actin polymerization in the invasion process. Microscopic analysis revealed various interactions, such as adherence, membrane alterations, vacuolization, apoptosis, and cellular damage. Anti-Omp34 serum-exposed A549 cells were protected and showed reduced damage. The findings reveal that A. baumannii can significantly multiply intracellularly within host cells. This suggests the bacterium's ability to establish an environment conducive to its replication by preventing fusion with degradative lysosomes and inhibiting acidification. This finding contributes to the understanding of A. baumannii's intracellular persistence and highlights the role of Omp34 in influencing apoptosis, autophagy, and bacterial adherence, which may impact the development of effective treatments against A. baumannii infections.

Beyond viability: Advancing CHO cell culture process strategies to modulate host cell protein levels

Chinese Hamster Ovary (CHO) cells are widely utilized in bioprocessing industry for monoclonal antibody (mAb) production. In many instances, challenges persist in achieving sufficient clearance of Host Cell Proteins (HCPs) in the final drug substance. While purification strategies usually offer substantial HCP clearance, certain "problematic" HCPs, particularly lipases, continue to pose significant challenges. This study investigates the accumulation of various "problematic" HCPs in CHO cell culture using transcriptomics, revealing correations between cell culture parameters and HCP level. Contrary to conventional assumptions, viability alone does not reliably predict HCP levels, with factors such as clone selection, host cell line choice, media and feed compositions significantly influencing HCP accumulation. Leveraging transcriptomics-based approaches, we demonstrate the potential of upstream process control strategies to mitigate HCP presence and improve biologic product quality. Our findings underscore the importance of considering diverse cell culture parameters in bioprocess optimization to ensure product stability and quality. While promising, further research is needed to elucidate the mechanisms underlying HCP release and propagation through downstream processing stages, emphasizing the necessity of a comprehensive integrated approach to HCP control in biologics production.

Recent Advancements in Proteomic Sample Preparation from Recombinant Chinese Hamster Ovary Cells.

Chinese hamster ovary (CHO) cells are the most commonly used mammalian host cell line for biopharmaceutical production because of their ability to correctly fold and post-translationally modify recombinant proteins that are compatible with human use. Proteomics, along with other "omic platforms," are being used to understand the biology of CHO cells with the ultimate aim to enhance CHO cell factories for more efficient production of biopharmaceuticals. Liquid chromatography-mass spectrometry (LC-MS) for proteomic analysis has become the standard technique for profiling proteomes. There are three stages to a typical bottom-up approach, namely, sample preparation, LC-MS/MS, and data analysis. Although there have been major advances in LC-MS/MS instrumentation and data analysis tools, sample preparation is still the crucial stage that affects the overall efficiency of any proteomic study. In this chapter, we present a comparison of a number of widely used sample preparation methods for proteomic applications, including in-solution digestion and commercially available device-based kits. All methods performed well; however, each method has inherent advantages and disadvantages.

Exploring P2X receptor activity: A journey from cellular impact to electrophysiological profiling

The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.

An autonucleolytic suspension HEK293F host cell line for high-titer serum-free AAV5 and AAV9 production with reduced levels of DNA impurity

We sought to engineer mammalian cells to secrete nuclease activity as a step toward removing the need to purchase commercial nucleases as process additions in bioprocessing of AAV5 and AAV9 as gene therapy vectors. Engineering HeLa cells with a serratial nuclease transgene did not bring about nuclease activity in surrounding media whereas engineering serum-free, suspension-adapted HEK293F cells with a staphylococcal nuclease transgene did result in detectable nuclease activity in surrounding media of the resultant stable transfectant cell line, "NuPro-1S." When cultivated in serum-free media, NuPro-1S cells yielded 3.06×1010 AAV5 viral genomes (vg)/mL via transient transfection, compared with 3.85×109 vg/mL from the parental HEK293F cell line. AAV9 production, followed by purification by ultracentrifugation, yielded 1.8×1013 vg/mL from NuPro-1S cells compared with 7.35×1012 vg/mL from HEK293F cells. AAV9 from both HEK293F and NuPro-1S showed almost identical ability to transduce cells embedded in a scaffold tissue mimic or cells of mouse neonate brain tissue invivo. Comparison of agarose gel data indicated that the DNA content of AAV5 and AAV9 process streams from NuPro-1S cells was reduced by approximately 60% compared with HEK293F cells. A similar reduction in HEK293F cells was only achievable with a 50U/mL Benzonase treatment.

CHO cell engineering via targeted integration of circular miR-21 decoy using CRISPR/RMCE hybrid system.

Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibitionof miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. KEY POINTS: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in.

Adeno-associated virus vector-based gene therapy for hereditary diseases: current problems of application and approaches to solve them

INTRODICTION. Currently, gene therapy based on adeno-associated virus (AAV) vectors faces a number of barriers, both biomedical and technological, which require studying and overcoming for further development of this gene therapy technology.AIM. This study aimed to analyse the use of gene therapy for a range of hereditary diseases, taking into account the barriers associated with its side effects and insufficient efficacy, the determination of the therapeutic window, and individual characteristics relevant to a particular hereditary disease; additionally, the study aimed to review the approaches to lifting these barriers and increasing the availability of gene therapy through the improvement of technological approaches to production and the reduction of production costs.DISCUSSION. The authors reviewed the experience accumulated for gene therapy products that were approved or undergoing clinical trials. The study included a gene therapy applicability assessment using several hereditary diseases as a case study. The assessment showed that correct determination of the therapeutic window for a medicinal product and timely diagnosis of a hereditary disease were essential for effective and safe gene therapy. The study considered the strategies used to reduce the risks of adverse events and increase the effectiveness of AAVbased gene therapy. The authors assessed technological advancements in the manufacturing of AAV-based gene therapy products. The most perspective directions were the transition to suspension culture systems, the improvement of bioreactors, the use of new methods and materials for the purification of viral particles, the improvement of transfection systems, and the creation of new host cell lines. Ultimately, this can lead to lower production costs and an increased availability of gene therapy.CONCLUSION. Currently, gene therapy is used only for a small range of hereditary diseases. Significant barriers to its use are due to insufficient efficacy, risks of adverse events, and high costs for treatment. Ongoing biomedical and technological development should lift many of these barriers and increase access to gene therapy.

RNASeq highlights ATF6 pathway regulators for CHO cell engineering with different impacts of ATF6β and WFS1 knockdown on fed-batch production of IgG1

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G1 (IgG1) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG1-producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity.

Enhancement of rAAV titers via inhibition of the interferon signaling cascade in transfected HEK293 suspension cultures.

The production of recombinant adeno-associated virus (rAAV) for gene therapy applications relies on the use of various host cell lines, with suspension-grown HEK293 cells being the preferred expression system due to their satisfactory rAAV yields in transient transfections. As the field of gene therapy continues to expand, there is a growing demand for efficient rAAV production, which has prompted efforts to optimize HEK293 cell line productivity through engineering. In contrast to other cell lines like CHO cells, the transcriptome of HEK293 cells during rAAV production has remained largely unexplored in terms of identifying molecular components that can enhance yields. In our previous research, we analyzed global regulatory pathways and mRNA expression patterns associated with increased rAAV production in HEK293 cells. Our data revealed substantial variations in the expression patterns between cell lines with low (LP) and high-production (HP) rates. Moving to a deeper layer for a more detailed analysis of inflammation-related transcriptome data, we detected an increased expression of interferon-related genes in low-producing cell lines. Following upon these results, we investigated the use of Ruxolitinib, an interferon pathway inhibitor, during the transient production of rAAV in HEK293 cells as potential media additive to boost rAAV titers. Indeed, we find a two-fold increase in rAAV titers compared to the control when the interferon pathways were inhibited. In essence, this work offers a rational design approach for optimization of HEK293 cell line productivity and potential engineering targets, ultimately paving the way for more cost-efficient and readily available gene therapies for patients.

Strategies to improve CHO cell culture performance: Targeted deletion of amino acid catabolism and apoptosis genes paired with growth inhibitor supplementation.

Chinese hamster ovary (CHO) cells are the predominant host of choice for recombinant monoclonal antibody (mAb) expression. Recent advancements in gene editing technology have enabled engineering new CHO hosts with higher growth, viability, or productivity. One approach involved knock out (KO) of BCAT1 gene, which codes for the first enzyme in the branched chain amino acid (BCAA) catabolism pathway; BCAT1 KO reduced accumulation of growth inhibitory short chain fatty acid (SCFA) byproducts and improved culture growth and titer when used in conjunction with high-end pH-controlled delivery of glucose (HiPDOG) technology and SCFA supplementation during production. Accumulation of SCFAs in the culture media is critical for metabolic shift toward higher specific productivity and hence titer. Here we describe knocking out BCKDHa/b genes (2XKO), which act downstream of the BCAT1, in a BAX/BAK KO CHO host cell line background to reduce accumulation of growth-inhibitory molecules in culture. Evaluation of the new 4XKO CHO cell lines in fed-batch production cultures (without HiPDOG) revealed that partial KO of BCKDHa/b genes in an apoptosis-resistant (BAX/BAK KO) background can achieve higher viabilities and mAb titers. This was evident when SCFAs were added to boost productivity as such additives negatively impacted culture viability in the WT but not BAX/BAK KO cells during batch production. Altogether, our findings suggest that SCFA addbacks can significantly increase productivity and mAb titers in the context of apoptosis-attenuated CHO cells with partial KO of BCAA genes. Such engineered CHO hosts can offer productivity advantages for expressing biotherapeutics in an industrial setting.

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.

Engineering an Autonucleolytic Mammalian Suspension Host Cell Line to Reduce DNA Impurity Levels in Serum-Free Lentiviral Process Streams.

We engineered HEK293T cells with a transgene encoding tetracycline-inducible expression of a Staphylococcus aureus nuclease incorporating a translocation signal. We adapted the unmodified and nuclease-engineered cell lines to grow in suspension in serum-free media, generating the HEK293TS and NuPro-2S cell lines, respectively. Transient transfection yielded 1.19 × 106 lentiviral transducing units per milliliter (TU/mL) from NuPro-2S cells and 1.45 × 106 TU/mL from HEK293TS cells. DNA ladder disappearance revealed medium-resident nuclease activity arising from NuPro-2S cells in a tetracycline-inducible manner. DNA impurity levels in lentiviral material arising from NuPro-2S and HEK293TS cells were undetectable by SYBR Safe agarose gel staining. Direct measurement by PicoGreen reagent revealed DNA to be present at 636 ng/mL in lentiviral material from HEK293TS cells, an impurity level reduced by 89% to 70 ng/mL in lentiviral material from NuPro-2S cells. This reduction was comparable to the 23 ng/mL achieved by treating HEK293TS-derived lentiviral material with 50 units/mL Benzonase.

Analysis of Host Cell Proteins in AAV Products with ProteoMiner Protein Enrichment Technology.

Despite substantial efforts to detect host cell proteins (HCPs) in antibody drugs, information regarding HCPs in gene therapy products remains limited and has not been widely integrated into the host cell engineering or purification processes. Most methods that have successfully detected HCPs in antibody drugs are not applicable to gene therapy products, except for the ProteoMiner enrichment method. Here, we demonstrate that ProteoMiner beads effectively enrich HCPs in adeno-associated virus (AAV) products and simultaneously remove the detergent Pluronic F-68 without a loss of low-abundance HCPs. Following optimization of this technique, there was up to a 34-fold increase in the enrichment of HCPs compared to direct digestion. Moreover, the detection limit was significantly lowered with the ability to detect HCPs at levels as low as 0.1 ng/mL after ProteoMiner treatment. This approach holds promise in AAV HCP analysis and may be adaptable to other gene therapy products. The findings from this study provide valuable insights into HCPs in AAV products and may facilitate process development and host cell line optimization. The high sensitivity of this approach also facilitates detection of critical low-abundance HCPs, thereby contributing to risk assessment of their impact on the safety and quality of the AAV-based gene therapy products.

Metatranscriptomic RNA-Seq Data Analysis of Virus-Infected Host Cells.

RNA sequencing (RNA-seq) analysis of virus-infected host cells enables researchers to study a wide range of phenomena involving host-virus interactions. This includes genomic analysis of the viral population itself, as well as analysis of the transcriptional dynamics of the virus and host during infection. In this chapter, we provide a guide for researchers interested in performing RNA-seq data analysis of virus-infected host cells or cell lines. We outline several bioinformatic protocols for quantifying viral abundance, assembling viral genomes from mixed samples, and performing differential expression analysis, among other common workflows. These workflows can be used as starting points for researchers aiming to analyze RNA-seq datasets of mixed samples containing both host and viral RNA, such as virus-infected cell lines or clinical samples.

#394 : The Efficacy of an Individualized Dose of Follitropin Delta on Ovary Reproductive Stimulation and Clinical Outcomes

Background and Aims: Follitropin delta is the third recombinant human follicle-stimulating hormone (r-hFSH) expressed in a host cell line of human fetal retinal origin that currently emphasizes that the actual tendency of administration is a personalized dosing algorithm based on the anti-Mullerian hormone (AMH) and body mass index (BMI) for ovarian stimulation. Methods: In this present manuscript, we aimed to gather all available data published between 2018–2022 regarding the co-administration and administration of follitropin delta and the clinical outcomes reported following an in vitro fertilization (IVF). Results: Follitropin delta is non-inferior in contrast to its previously launched agents for ovarian stimulation, enhancing a similar-to-superior response reflected by both the reproductive and pregnancy outcomes in parallel with a low risk of ovarian hyperstimulation syndrome (OHSS), being well tolerated. The body weight and AMH level are factors that may influence the outcome in a patient. Despite controversy and results that refute these arguments on several occasions, follitropin delta exceeds the benefits of conventional dosing with either follitropin alfa or follitropin beta. Therefore, all post hoc derived analyzes and subgroups of patients who participated in subsequent studies support this statement. Conclusion: Despite the relatively limited range of data in the current literature, most authors provide strong evidence to support the later use of this drug according to patient profiles and overcoming ethnic limitations. Others disagree with these observations, but this topic and this drug have great potential. As such, additional research is needed to fill existing knowledge gaps and extend this experience to a larger scale. Demonstrate overcoming all limitations.

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Chinese hamster ovary (CHO) cells are the cell line of choice for producing recombinant therapeutic proteins. Despite improvements in production processes, reducing manufacturing costs remains a key driver in the search for more productive clones. To identify media additives capable of increasing protein production, CHOZN® GS-/- cell lines were screened with 1280 small molecules, and two were identified, forskolin and BrdU, which increased productivity by ≥40%. While it is possible to incorporate these small molecules into a commercial-scale process, doing so may not be financially feasible or could raise regulatory concerns related to the purity of the final drug substance. To circumvent these issues, RNA-Seq was performed to identify transcripts which were up- or downregulated upon BrdU treatment. Subsequent Reactome pathway analysis identified the electron transport chain as an affected pathway. CRISPR/Cas9 was utilized to create missense mutations in two independent components of the electron transport chain and the resultant clones partially recapitulated the phenotypes observed upon BrdU treatment, including the productivity of recombinant therapeutic proteins. Together, this work suggests that BrdU can enhance the productivity of CHO cells by modulating cellular energetics and provides a blueprint for translating data from small molecule chemical screens into genetic engineering targets to improve the performance of CHO cells. This could ultimately lead to more productive host cell lines and a more cost-effective method of supplying medication to patients.

Efficient showering vaccination with a live attenuated vaccine against herpesviral hematopoietic necrosis in goldfish

Herpesviral hematopoietic necrosis caused by cyprinid herpesvirus 2 (CyHV-2) frequently affects farmed goldfish Carassius auratus and gibel carp C. auratus gibelio, leading to significant economic damage. In our previous study, a live attenuated vaccine, called the P7-P8 vaccine strain, was developed by propagating virulent CyHV-2 in two non-natural host cell lines. The vaccine demonstrated high protective efficacy in goldfish with no signs of virulence reversion. Methods that reduce the costs of the procedure, including labor costs, are warranted for the practical application of this vaccine in goldfish aquaculture. The aim of this study was to explore alternative vaccination protocols for the P7-P8 strain in goldfish. First, we tested the previous standard administration protocol which is the bath vaccination with 1: 1000 diluted vaccine cultures for 2 h. In the simple first step to reduce vaccine virus culture, we examined the bathing protocol with more-diluted virus culture (ranging from 1: 1000 to 1: 81,000). The relative percentage survival (RPS) after the virulent virus immersion challenge for 2 h at 21 days post-vaccination was remarkably decreased in a dose-dependent manner; a dilution of 1: 3000 resulted in a 19% reduction from that used in the standard bathing protocol. Therefore, alternative methods (dipping and showering vaccination protocols) were tested using a 1: 100 dilution vaccine culture (10-fold more concentrated than the standard protocol) with short exposure times. Dipping vaccination for 10 s, 1 min, 2 min, and 5 min showed sufficient RPS of 82.4%–100%. The 10 s process was repeated six times using the same vaccine solution, resulting in an RPS >89%. In the showering vaccination protocol, the vaccine solution (1: 100 dilution; 1 mL, 3 mL, or 5 mL) was showered onto 20 goldfish on a net and held for 10 s, resulting in an RPS of >89%. We selected the showering vaccination as the suitable vaccination protocol. The effectiveness of this vaccination protocol was tested using the virus challenge by employing the cohabitation method with infected fish. The results showed high protective efficacy with an RPS of 65%–100%. This study demonstrated that the showering vaccination protocol, which uses small amounts of vaccine culture and produces less waste vaccine solution with short labor time, may be considered as a suitable alternative to the standard bath method in goldfish aquaculture.