Gene copy number, gene configuration and LC/HC mRNA ratio impact on antibody productivity and product quality in targeted integration CHO cell lines.

The augmentation of transgene copy numbers is a prevalent approach presumed to enhance transcriptional activity and product yield. CHO cell lines engineered via targeted integration (TI) offer an advantageous platform for investigating the interplay between gene copy number, mRNA abundance, product yield, and product quality. Our investigation revealed that incrementally elevating the gene copy numbers of both IgG heavy chain (HC) and light chain (LC) concurrently resulted in the attainment of plateaus in mRNA levels and product titers, notably occurring beyond four to five gene copies integrated at the same TI site. Furthermore, maintaining a fixed gene copy number while varying the position of genes within the vector influenced the LC/HC mRNA ratio, which subsequently exerted a substantial impact on product titer. Moreover, manipulation of the LC/HC gene ratio through the introduction of surplus LC gene copies led to heightened LC mRNA expression and a reduction in the levels of high molecular weight species. It is noteworthy that the effects of excess LC on product titer were dependent on the specific molecule under consideration. The strategic utilization of PCR tags enabled precise quantification of transcription from each expression slot within the vector, facilitating the identification of highly expressive and less expressive slots. Collectively, these findings significantly enhance our understanding of stable antibody production in TI CHO cell lines.

Multi-omics kinetic analysis of recombinant adeno-associated virus production by plasmid transfection of HEK293 cells.

Recombinant adeno-associated virus (rAAV) is among the most commonly used vectors for gene therapy. It is commonly produced by transfection of HEK293 cells with three plasmids each containing the vector genome including gene of interest (GOI), helper functions, and rep and cap genes for genome replication and capsid formation. To meet the potential clinical needs, the productivity of the production system needs to be enhanced. A better process characterization of the production system will further advance our insights into ways to enhance productivity. Here, we employed transcriptomic analysis to quantify the dynamics of different isoforms of viral transcripts and to assess the shift of cellular physiology, and deployed targeted proteomic analysis for absolute quantification of viral proteins and tandem mass tags (TMTs) for assessing cellular responses at the protein level. Functional analysis at transcriptome and proteome levels identified defense and immune response, unfolded protein response, p53 signaling as enriched. The small molecule additive intervention study based on functional analysis showed the potential of such omics-guided productivity enhancement. Together, multi-omics analysis advanced understanding of rAAV production and provided insight into enhancing rAAV production by plasmid transfection.

Enhancing the production of recombinant adeno-associated virus in synthetic cell lines through systematic characterization.

Recombinant adeno-associated virus (rAAV) is among the most commonly used in vivo gene delivery vehicles and has seen a number of successes in clinical application. Current manufacturing processes of rAAV employ multiple plasmid transfection or rely on virus infection and face challenges in scale-up. A synthetic biology approach was taken to generate stable cell lines with integrated genetic modules, which produced rAAV upon induction albeit at a low productivity. To identify potential factors that restrained the productivity, we systematically characterized virus production kinetics through targeted quantitative proteomics and various physical assays of viral components. We demonstrated that reducing the excessive expression of gene of interest by its conditional expression greatly increased the productivity of these synthetic cell lines. Further enhancement was gained by optimizing induction profiles and alleviating proteasomal degradation of viral capsid protein by the addition of proteasome inhibitors. Altogether, these enhancements brought the productivity close to traditional multiple plasmid transfection. The rAAV produced had comparable full particle contents as those produced by conventional transient plasmid transfection. The present work exemplified the versatility of our synthetic biology-based viral vector production platform and its potential for plasmid- and virus-free rAAV manufacturing.

Regulation of Flowering Time by Environmental Factors in Plants.

The timing of floral transition is determined by both endogenous molecular pathways and external environmental conditions. Among these environmental conditions, photoperiod acts as a cue to regulate the timing of flowering in response to seasonal changes. Additionally, it has become clear that various environmental factors also control the timing of floral transition. Environmental factor acts as either a positive or negative signal to modulate the timing of flowering, thereby establishing the optimal flowering time to maximize the reproductive success of plants. This review aims to summarize the effects of environmental factors such as photoperiod, light intensity, temperature changes, vernalization, drought, and salinity on the regulation of flowering time in plants, as well as to further explain the molecular mechanisms that link environmental factors to the internal flowering time regulation pathway.

The C. elegans deglutamylase CCPP-6 does not operate redundantly with CCPP-1 in gross cilia integrity.

Tubulin glutamylation is a reversible modification of the microtubules that regulates cilia stability and function. The addition of glutamates to the microtubule is catalyzed by members of the TTLL family of enzymes, while the removal is carried out by a family of cytosolic carboxypeptidase (CCP) enzymes. C. elegans has two deglutamylating enzymes, CCPP-1 and CCPP-6 . CCPP-1 is required for ciliary stability and function in the worm, however CCPP-6 is dispensable for cilia integrity. To investigate redundancy between the two deglutamylating enzymes we made a ccpp-1 ( ok1821 ); ccpp-6 ( ok382 ) double mutant. The double mutant shows normal viability, and the dye-filling phenotypes are not worse than the ccpp-1 single mutant, suggesting that CCPP-1 and CCPP-6 do not function redundantly in C. elegans cilia .

Modulation of lignin biosynthesis for drought tolerance in plants.

Lignin is a complex polymer that is embedded in plant cell walls to provide physical support and water protection. For these reasons, the production of lignin is closely linked with plant adaptation to terrestrial regions. In response to developmental cues and external environmental conditions, plants use an elaborate regulatory network to determine the timing and location of lignin biosynthesis. In this review, we summarize the canonical lignin biosynthetic pathway and transcriptional regulatory network of lignin biosynthesis, consisting of NAC and MYB transcription factors, to explain how plants regulate lignin deposition under drought stress. Moreover, we discuss how the transcriptional network can be applied to the development of drought tolerant plants.

Signaling pathways underlying nitrogen transport and metabolism in plants.

Nitrogen (N) is an essential macronutrient required for plant growth and crop production. However, N in soil is usually insufficient for plant growth. Thus, chemical N fertilizer has been extensively used to increase crop production. Due to negative effects of N rich fertilizer on the environment, improving N usage has been a major issue in the field of plant science to achieve sustainable production of crops. For that reason, many efforts have been made to elucidate how plants regulate N uptake and utilization according to their surrounding habitat over the last 30 years. Here, we provide recent advances focusing on regulation of N uptake, allocation of N by N transporting system, and signaling pathway controlling N responses in plants. [BMB Reports 2023; 56(2): 56-64].

Development of an Inducible, Replication-Competent Assay Cell Line for Titration of Infectious Recombinant Adeno-Associated Virus Vectors.

An important quality attribute of a recombinant adeno-associated virus (rAAV) as a therapeutic vector is its infectivity. Current assays to quantify infectious rAAV rely on coinfection with a helper virus such as adenovirus (Ad), which requires helper virus preparation and introduces additional variability. A simple method that has high sensitivity and removes the need for helper virus would improve assay consistency and facilitate high-throughput applications such as rAAV producer cell line development. In this study, we describe a stable assay cell line that was generated by integrating the coding sequences for AAV Rep68 and Ad E4orf6 and DNA binding protein under the control of inducible promoters. The Rep68 protein expression was further modulated by a ligand-responsive destabilization domain. In several benchmarks, the cell line gave comparable titers with those obtained using a classical Ad coinfection method. The cell line was also used to titer vectors of multiple rAAV serotypes. This cell line has the potential to serve as an effective and robust tool for quantifying infectious rAAV titers to advance gene therapy vector biomanufacturing.

Construction of an rAAV Producer Cell Line through Synthetic Biology.

Recombinant adeno-associated viruses (rAAV) are important gene delivery vehicles for gene therapy applications. Their production relies on plasmid transfection or virus infection of producer cells, which pose a challenge in process scale-up. Here, we describe a template for a transfection-free, helper virus-free rAAV producer cell line using a synthetic biology approach. Three modules were integrated into HEK293 cells including an rAAV genome and multiple inducible promoters controlling the expression of AAV Rep, Cap, and helper coding sequences. The synthetic cell line generated infectious rAAV vectors upon induction. Independent control over replication and packaging activities allowed for manipulation of the fraction of capsid particles containing viral genomes, affirming the feasibility of tuning gene expression profiles in a synthetic cell line for enhancing the quality of the viral vector produced. The synthetic biology approach for rAAV production presented in this study can be exploited for scalable biomanufacturing.

Segment-specific kinetics of mRNA, cRNA and vRNA accumulation during influenza infection.

Influenza A virus (IAV) is a segmented negative-sense RNA virus and is the cause of major epidemics and pandemics. The replication of IAV is complex, involving the production of three distinct RNA species; mRNA, cRNA, and vRNA for all eight genome segments. While understanding IAV replication kinetics is important for drug development and improving vaccine production, current methods for studying IAV kinetics has been limited by the ability to detect all three different RNA species in a scalable manner. Here we report the development of a novel pipeline using total stranded RNA-Seq, which we named Influenza Virus Enumerator of RNA Transcripts (InVERT), that allows for the simultaneous quantification of all three RNA species produced by IAV. Using InVERT, we provide a full landscape of the IAV replication kinetics and found that different groups of viral genes follow different kinetics. The segments coding for RNA-dependent RNA Polymerase (RdRP) produced more vRNA than mRNA while some other segments (NP, NS, HA) consistently made more mRNA than vRNA. vRNA expression levels did not correlate with cRNA expression, suggesting complex regulation of vRNA synthesis. Furthermore, by studying the kinetics of a virus lacking the capacity to generate new polymerase complexes, we found evidence that further supports the model that cRNA synthesis requires newly synthesized RdRP and that incoming RdRP can only generate mRNA. Overall, InVERT is a powerful tool for quantifying IAV RNA species to elucidate key features of IAV replication.ImportanceInfluenza A virus (IAV) is a respiratory pathogen that has caused significant mortality throughout history and remains a global threat to human health. Although much is known about IAV replication, the regulation of IAV replication dynamics is not completely understood. This is due in part to both technical limitations and the complexity of the virus replication, which has a segmented genome and produces three distinct RNA species for each gene segment. We developed a new approach that allows the methodical study of IAV replication kinetics, shedding light on many interesting features of IAV replication biology. This study advances our understanding of the kinetics of IAV replication and will help to facilitate future research in the field.

Epigenomic features revealed by ATAC-seq impact transgene expression in CHO cells.

Different regions of a mammalian genome have different accessibilities to transcriptional machinery. The integration site of a transgene affects how actively it is transcribed. Highly accessible genomic regions called super-enhancers have been recently described as strong regulatory elements that shape cell identity. Super-enhancers have been identified in Chinese hamster ovary (CHO) cells using the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq). Genes near super-enhancer regions had high transcript levels and were enriched for oncogenic signaling and proliferation functions, consistent with an immortalized phenotype. Inaccessible regions in the genome with low ATAC signal also had low transcriptional activity. Genes in inaccessible regions were enriched for remote tissue functions such as taste, smell, and neuronal activation. A lentiviral reporter integration assay showed integration into super-enhancer regions conferred higher reporter expression than insertion into inaccessible regions. Targeted integration of an IgG vector into the Plec super-enhancer region yielded clones that expressed the immunoglobulin light chain gene mostly in the top 20% of all transcripts with the majority in the top 5%. The results suggest the epigenomic landscape of CHO cells can guide the selection of integration sites in the development of cell lines for therapeutic protein production.

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

For the biomanufacturing of protein biologics, establishing stable cell lines with high transgene transcription is critical for high productivity. Modern genome engineering tools can direct transgene insertion to a specified genomic locus and can potentially become a valuable tool for cell line generation. In this study, the authors survey transgene integration sites and their transcriptional activity to identify characteristics of desirable regions. A lentivirus containing destabilized Green Fluorescent Protein (dGFP) is used to infect Chinese hamster ovary cells at a low multiplicity of infection, and cells with high or low GFP fluorescence are isolated. RNA sequencing and Assay for Transposase Accessible Chromatin using sequencing data shows integration sites with high GFP expression are in larger regions of high transcriptional activity and accessibility, but not necessarily within highly transcribed genes. This method is used to obtain high Immunoglobulin G (IgG) expressing cell lines with a single copy of the transgene integrated into transcriptionally active and accessible genomic regions. Dual recombinase-mediated cassette exchange is then employed to swap the IgG transgene for erythropoietin or tumor necrosis factor receptor-Fc. This work thus highlights a strategy to identify desirable sites for transgene integration and to streamline the development of new product producing cell lines.

Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer.

The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes-which express particularly high levels of GLUT1-have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells.