As the demand for monoclonal antibodies (mAb) increases, more efficient expression methods are required for their manufacturing process. Transcriptional gene silencing is a common phenomenon in recombinant cell lines which leads to expression reduction and instability. There are reports on improved antibody expression in ubiquitous chromatin opening element (UCOE) containing both heavy and light chain gene constructs. Here we investigate the impact of having these elements as part of the light chain, heavy chain or both genes during cell line development. In this regard, non-UCOE and UCOE vectors were constructed and stable Chinese hamster ovary (CHO) cell pools were generated by different vector combinations. Expression analysis revealed that all UCOE cell pools had higher antibody yields compared to non-UCOE cells, Moreover the most optimal expression was obtained by cells containing just the UCOE on heavy chain. In terms of stability, it was shown that the high level of expression was kept consistence for more than four months in these cells whereas the expression titers were reduced in the other UCOE pools. In conclusion, UCOE significantly enhanced the level and stability of antibody expression and the use of this element with heavy chain provided more stable cell lines with higher production level.

The optimization of bioprocess conditions towards improved growth profile and productivity yield is considered of great importance in biopharmaceutical manufacturing. Peptones as efficient sources of nutrients have been studied for their effect on media development; however, their role on metabolic pathway is not well understood. In the present study, the effect of different concentration of peptones on a recombinant Chinese hamster ovary (CHO) cell line grown in three serum-free suspension cultures was determined. Six peptones of different origins and available amino acid profiles were investigated regarding their impact on cell growth, productivity, and metabolic pathways changes. In optimized feeding strategies, increases of 136% and 159% in volumetric productivity (for a low-nutrient culture media) and 55% (for a high-nutrient culture media) were achieved. Furthermore, particular sources of peptones with specific amino acid profile developed preferential results for each different culture medium. Two peptones, SoyA2SC and SoyE-110, were the only hydrolysates that showed production improvement in all three media. Casein Peptone plus Tryptone N1 and SoyA3SC showed different improved results based on their implemented concentration for each individual basal medium. The amino acid profile of peptones may provide clues to identify the most effective feeding strategies for recombinant CHO cells.

In recent years, monoclonal antibodies (mAbs) have been developed as powerful therapeutic and diagnostic agents and Chinese hamster ovary (CHO) cells have emerged as the dominant host for the recombinant expression of these proteins. A critical step in recombinant expression is the utilization of strong promoters, such as the Chinese Hamster Elongation Factor-1α (CHEF-1) promoter. To compare the strengths of CHEF with cytomegalovirus (CMV) promoter for mAb expression in CHO cells, four bicistronic vectors bearing either internal ribosome entry site (IRES) or Furin/2A (F2A) sequences were designed. The efficiency of these promoters was evaluated by measuring level of expressed antibody in stable cell pools. Our results indicated that CHEF promoter-based expression of mAbs was 2.5 fold higher than CMV-based expression in F2A-mediated vectors. However, this difference was less significant in IRES-mediated mAb expressing cells. Studying the stability of the F2A expression system in the course of 18 weeks, we observed that the cells having CHEF promoter maintained their antibody expression at higher level than those transfected with CMV promoter. Further analyses showed that both IRES-mediated vectors, expressed mAbs with correct size, whereas in antibodies expressed via F2A system heterogeneity of light chains were detected due to incomplete furin cleavage. Our findings indicated that the CHEF promoter is a viable alternative to CMV promoter-based expression in F2A-mediated vectors by providing both higher expression and level of stability.

Pseudomonas aeruginosa(PA) is a leading cause of nosocomial infections and death in cystic fibrosis patients. The study was conducted to evaluate the physicochemical structure, biological activity and serum stability of a recombinant anti-PcrV single chain variable antibody fragment genetically attached to the mCH3cc domain. The stereochemical properties of scFv-mCH3 (YFL001) and scFv (YFL002) proteins as well as molecular interactions towardsPseudomonas aeruginosaPcrV were evaluated computationally. The subcloned fragments encoding YFL001 and YFL002 in pET28a were expressed within theE. coliBL21-DE3 strain. After Ni–NTA affinity chromatography, the biological activity of the proteins in inhibition of PA induced hemolysis as well as cellular cytotoxicity was assessed. In silico analysis revealed the satisfactory stereochemical quality of the models as well as common residues in their interface with PcrV. The structural differences of proteins through circular dichroism spectroscopy were confirmed by NMR analysis. Both proteins indicated inhibition of ExoU positive PA strains in hemolysis of red blood cells compared to ExoU negative strains as well as cytotoxicity effect on lung epithelial cells. The ELISA test showed the longer serum stability of the YFL001 molecule than YFL002. The results were encouraging to further evaluation of these two scFv molecules in animal models.

Pancreatic cancer is marked by a poor prognosis and an exceptionally high mortality rate, with its aggressive nature contributing to its classification as a highly malignant disease. For effective therapeutic strategies, the development of sophisticated and regulated DNA manipulation methods is essential. Originally part of the prokaryotic immune system, CRISPR/Cas has emerged as a pivotal genome-editing tool with promising applications in pancreatic cancer research and therapy. This gene editing method is known for simplicity, rapid advancement, and superior precision compared to earlier techniques. Its adaptability allows precise gene editing for therapeutic purposes, including oncogene silencing and correction of pathogenic mutations. Additionally, CRISPR-driven gene editing has facilitated the development of pancreatic cancer models, which serve as valuable platforms for drug discovery and personalized treatment strategies, offering deeper insights into the genetic landscape of pancreatic tumors. This article provides an overview of the current applications of CRISPR technology in gene therapy and cancer research, particularly in the context of pancreatic cancer, and lays the foundation for future studies.