Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells.

With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells.

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.

In vitro assessment of the risk of ABCB1-mediated drug-drug interaction between rivaroxaban and tacrolimus in human embryonic kidney 293 recombinant cell lines.

Background: In lung transplant patients, direct oral anticoagulants are often taken in combination with immunosuppressive drugs such as tacrolimus. Since tacrolimus is a substrate and inhibitor of the efflux protein ABCB1, also transporting direct oral anticoagulants, a possible drug-drug interaction mediated by competition for this transporter needs to be investigated. Objectives: To determine the in vitro effect of tacrolimus on ABCB1-mediated rivaroxaban transport in order to support clinician practice. Methods: Recombinant cell line models, based on human embryonic kidney 293 cells, were generated by a stable transfection process to overexpress ABCB1 or not (control cells). The impact of tacrolimus on ABCB1-mediated rivaroxaban transport was assessed by accumulation experiments. Results: ABCB1 expression decreased the cellular accumulation of rivaroxaban and tacrolimus at their respective clinically relevant concentrations when compared with control cells. This confirms the involvement of ABCB1 in the active transport of tacrolimus and rivaroxaban. However, tacrolimus had no significant influence on rivaroxaban disposition at those clinically relevant concentrations. Conclusion: Our study does not provide evidence for a possible interaction between tacrolimus and rivaroxaban when used together in practice.

α-Lipoic acid increases the viability of nephrocytes and elevates sulfane sulfur level in plasma of patients with chronic kidney disease.

BACKGROUND: Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. METHODS: The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. RESULTS: The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.

Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells.

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Antioxidative and anti-cytogenotoxic potential of Lysiphyllum strychnifolium (Craib) A. Schmitz extracts against cadmium-induced toxicity in human embryonic kidney (HEK293) and dermal fibroblast (HDF) cells.

Exposure to cadmium (Cd) results in bioaccumulation and irreversible damage; this encourages an investigation of alternatives to address Cd toxicity, using natural compounds. Lysiphyllum strychnifolium, a well-known Thai medicinal plant, was investigated for its phytochemical compounds and corresponding bioactivities, including antioxidant and anti-cytogenotoxic effects against Cd toxicity in HEK293 renal and HDF dermal cell models. The crude extract of L. strychnifolium (LsCrude) was partitioned into four fractions, using sequential polarity solvents (hexane, dichloromethane, ethyl acetate, and water, denoted as LsH, LsD, LsE, and LsW, respectively). The extraction yields were 1.79 %, 5.08 %, 8.53 %, and 70.25 % (w/w), respectively. Phytochemical screening revealed the presence of tannins, alkaloids, and flavonoids in LsCrude and its fractions, except for LsH. LsE exhibited the highest concentrations of phenolics (286.83 ± 6.83 mg GAE/g extract) and flavonoids (86.36 ± 1.29 mg QE/g extract). Subsequent 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and ferric-reducing ability of plasma (FRAP) reducing powder assays demonstrated the high antioxidant capacity of LsCrude and its fractions. The lowest IC50 value (9.11 ± 0.43 µg/mL) in the DPPH assay corresponded to LsW, whereas the highest total FRAP value (6.06 ± 0.70 mg QE Eq./g dry mass) corresponded to LsE. MTT and alkaline comet assays revealed the lack of toxicity of the extracts, which were considered safe. Upon exposure to Cd at the CC50 level, HEK293 cells treated with LsE suppressed Cd-induced damage. HDF cells treated with LsCrude, LsD, or LsE attenuated Cd-induced damage. In the pre-treatment, LsD protected the HDF cells against Cd-mediated cytogenotoxicity. These anti-cytogenotoxic potentials are likely due to the antioxidant properties of the phytochemicals. Our findings highlight the cyto-geno-protective properties of L. strychnifolium stem extracts against Cd toxicity in HEK293 and HDF cells, and provide a novel approach for combating oxidative stress and DNA damage caused by environmental pollutants.

Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.

Hypoxia decreases mitochondrial ROS production in cells.

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells' ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

Changes in the Transcriptome and Long Non-Coding RNAs but Not the Methylome Occur in Human Cells Exposed to Borrelia burgdorferi.

Lyme disease, caused by infection with members of the Lyme borreliosis group of Borrelia spirochete bacteria, is increasing in frequency and distribution worldwide. Epigenetic interactions between the mammalian host, tick, and bacterial pathogen are poorly understood. In this study, high-throughput next-generation sequencing (NGS) allowed for the in vitro study of the transcriptome, non-coding RNAs, and methylome in human host cells in response to Borrelia burgdorferi infection. We tested the effect of the Borrelia burgdorferi strain B31 on a human primary cell line (HUVEC) and an immortalized cell line (HEK-293) for 72 h, a long-duration time that might allow for epigenetic responses in the exposed human host cells. Differential gene expression was detected in both cell models in response to B. burgdorferi. More differentially expressed genes were found in HUVECs compared to HEK-293 cells. Borrelia burgdorferi exposure significantly induced genes in the interferon, in addition to cytokine and other immune response signaling in HUVECs. In HEK-293 cells, pre-NOTCH processing in Golgi was significantly downregulated in Borrelia-exposed cells. Other significantly altered gene expressions were found in genes involved in the extracellular matrix. No significant global methylation changes were detected in HUVECs or HEK-293 cells exposed to B. burgdorferi; however, two long non-coding RNAs and a pseudogene were deregulated in response to B. burgdorferi in HUVECs, suggesting that other epigenetic mechanisms may be initiated by infection.

Zelquistinel acts at an extracellular binding domain to modulate intracellular calcium inactivation of N-methyl-d-aspartate receptors.

Stinels are a novel class of N-methyl-d-aspartate glutamate receptor (NMDAR) positive allosteric modulators. We explored mechanism of action and NR2 subtype specificity of the stinel zelquistinel (ZEL) in HEK 293 cells expressing recombinant NMDARs. ZEL potently enhanced NMDAR current at NR2A (EC50 = 9.9 ± 0.5 nM) and NR2C-containing (EC50 = 9.7 ± 0.6 nM) NMDARs, with a larger ceiling enhancement at NR2B-NMDAR (EC50 = 35.0 ± 0.7 nM), while not affecting NR2D-containing NMDARs. In cells expressing NR2A and NR2C-containing NMDARs, ZEL exhibited an inverted-U dose-response relation, with a low concentration enhancement and high concentration suppression of NMDAR currents. Extracellular application of ZEL potentiated NMDAR receptor activity via prolongation of NMDAR currents. Replacing the slow Ca2+ intracellular chelator EGTA with the fast chelator BAPTA blocked ZEL potentiation of NMDARs, suggesting an action on intracellular Ca2+-calmodulin-dependent inactivation (CDI). Consistent with this mechanism of action, removal of the NR1 intracellular C-terminus, or intracellular infusion of a calmodulin blocking peptide, blocked ZEL potentiation of NMDAR current. In contrast, BAPTA did not prevent high-dose suppression of current, indicating this effect has a different mechanism of action. These data indicate ZEL is a novel positive allosteric modulator that binds extracellularly and acts through a unique long-distance mechanism to reduce NMDAR CDI, eliciting enhancement of NMDAR current. The critical role that NMDARs play in long-term, activity-dependent synaptic plasticity, learning, memory and cognition, suggests dysregulation of CDI may contribute to psychiatric disorders such as depression, schizophrenia and others, and that the stinel class of drugs can restore NMDAR-dependent synaptic plasticity by reducing activity-dependent CDI.

In silico, in vitro, and in vivo acute and sub-acute toxicity profiling of whole plant methanol extract of Equisetum diffusum D. Don from the sub-Himalayan West Bengal, India, having ethnobotanical uses.

BACKGROUND: Equisetum diffusum D. Don commonly known as 'Himalayan horsetail', has been traditionally used in the treatment of back pain, bone fracture and dislocation, and arthritis by various tribal communities of India. Our previous study confirmed the anti-inflammatory efficacy of the plant through in silico, in vitro, and in vivo model studies. Therefore, the current research is focused on safety dose evaluation for the first-time of the whole-plant methanol extract (EDME) of E. diffusum through appropriate in silico, in vitro, and in vivo approaches. METHOD: The whole plant, along with its rhizomes, was collected, and the methanol extract was prepared. The in silico ADMET study was performed to predict the pharmacokinetics profile and toxicity of all the identified phyto-compounds of EDME previously screened by GC-MS study. In vitro cytotoxicity study of EDME was performed using two cell lines: kidney (HEK293) and liver (Huh7) cell lines. The in vivo toxicity study of EDME was validated by the acute toxicity (OECD 423, 2002) and sub-acute toxicity assays (OECD 407, 2008) in the Wistar Albino rat model. RESULTS: The in silico ADMET study of all 47 bioactives predicted good pharmacokinetic and low toxicity profiles. In vitro cytotoxicity showed higher IC50 values of EDME viz., 672 ± 15.7 µg/mL and 1698 ± 6.54 µg/mL for both kidney (HEK293) and liver (Huh7) cell lines, respectively, which were considered as low-toxic. Based on acute oral toxicity, the LD50 value of the extract was considered "non-toxic" up to a feeding range of 2000 mg/kg of body weight. The regular consumption of the extract for an extended period (28 days) was also qualified as safe based on the body and organ weight, hematological, biochemical, and histoarchitecture results in the sub-acute toxicity assay. CONCLUSION: The detailed in silico, in vitro, in vivo (acute and sub-acute oral toxicity) studies gave us a new insight to the safety dose evaluation of Equisetum diffusum, which may serve as a reliable documentation for undertaking the experimental validation of the ethnobotanical uses of the plant which would help in the field of drug development for the treatment of inflammation related complications.

High-yield extracellular vesicle production from HEK293T cells encapsulated in 3D auxetic scaffolds with cyclic mechanical stimulation for effective drug carrier systems.

Extracellular vesicles (EVs) show promise in drug loading and delivery for medical applications. However, the lack of scalable manufacturing processes hinders the generation of clinically suitable quantities, thereby impeding the translation of EV-based therapies. Current EV production relies heavily on non-physiological two-dimensional (2D) cell culture or bioreactors, requiring significant resources. Additionally, EV-derived ribonucleic acid cargo in three-dimensional (3D) and 2D culture environments remains largely unknown. In this study, we optimized the biofabrication of 3D auxetic scaffolds encapsulated with human embryonic kidney 293 T (HEK293 T) cells, focusing on enhancing the mechanical properties of the scaffolds to significantly boost EV production through tensile stimulation in bioreactors. The proposed platform increased EV yields approximately 115-fold compared to conventional 2D culture, possessing properties that inhibit tumor progression. Further mechanistic examinations revealed that this effect was mediated by the mechanosensitivity of YAP/TAZ. EVs derived from tensile-stimulated HEK293 T cells on 3D auxetic scaffolds demonstrated superior capability for loading doxorubicin compared to their 2D counterparts for cancer therapy. Our results underscore the potential of this strategy for scaling up EV production and optimizing functional performance for clinical translation.

Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.

Two-Step Enrichment Facilitates Background Reduction for Proteomic Analysis of Lysosomes.

Lysosomes constitute the main degradative compartment of most mammalian cells and are involved in various cellular functions. Most of them are catalyzed by lysosomal proteins, which typically are low abundant, complicating their analysis by mass spectrometry-based proteomics. To increase analytical performance and to enable profiling of lysosomal content, lysosomes are often enriched. Two approaches have gained popularity in recent years, namely, superparamagnetic iron oxide nanoparticles (SPIONs) and immunoprecipitation from cells overexpressing a 3xHA-tagged version of TMEM192 (TMEM-IP). The effect of these approaches on the lysosomal proteome has not been investigated to date. We addressed this topic through a combination of both techniques and proteomic analysis of lysosome-enriched fractions. For SPIONs treatment, we identified altered cellular iron homeostasis and moderate changes of the lysosomal proteome. For overexpression of TMEM192, we observed more pronounced effects in lysosomal protein expression, especially for lysosomal membrane proteins and those involved in protein trafficking. Furthermore, we established a combined strategy based on the sequential enrichment of lysosomes with SPIONs and TMEM-IP. This enabled increased purity of lysosome-enriched fractions and, through TMEM-IP-based lysosome enrichment from SPIONs flow-through and eluate fractions, additional insights into the properties of individual approaches. All data are available via ProteomeXchange with PXD048696.

CRISPR Base Editing to Create Potential Charcot-Marie-Tooth Disease Models with High Editing Efficiency: Human Induced Pluripotent Stem Cell Harboring SH3TC2 Variants.

Human induced pluripotent stem cells (hiPSCs) represent a powerful tool to investigate neuropathological disorders in which the cells of interest are inaccessible, such as in the Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy. Developing appropriate cellular models becomes crucial in order to both study the disease's pathophysiology and test new therapeutic approaches. The generation of hiPS cellular models for disorders caused by a single nucleotide variation has been significantly improved following the development of CRISPR-based editing tools. In this study, we efficiently and quickly generated, by CRISPR editing, the two first hiPSCs cellular models carrying alterations involved in CMT4C, also called AR-CMTde-SH3TC2. This subtype of CMT is associated with alterations in the SH3TC2 gene and represents the most prevalent form of autosomal recessive demyelinating CMT. We aimed to develop models for two different SH3TC2 nonsense variants, c.211C>T, p.Gln71* and the most common AR-CMTde-SH3TC2 alteration, c.2860C>T, p.Arg954*. First, in order to determine the best CRISPR strategy to adopt on hiPSCs, we first tested a variety of sgRNAs combined with a selection of recent base editors using the conveniently cultivable and transfectable HEK-293T cell line. The chosen CRISPR base-editing strategy was then applied to hiPSCs derived from healthy individuals to generate isogenic CMT disease models with up to 93% editing efficiency. For point mutation generation, we first recommend to test your strategies on alternative cell line such as HEK-293T before hiPSCs to evaluate a variety of sgRNA-BE combinations, thus boosting the chance of achieving edited cellular clones with the hard-to-culture and to transfect hiPSCs.

In vitro screening of understudied PFAS with a focus on lipid metabolism disruption.

Per- and polyfluoroalkyl substances (PFAS) are man-made chemicals used in many industrial applications. Exposure to PFAS is associated with several health risks, including a decrease in infant birth weight, hepatoxicity, disruption of lipid metabolism, and decreased immune response. We used the in vitro cell models to screen six less studied PFAS [perfluorooctane sulfonamide (PFOSA), perfluoropentanoic acid (PFPeA), perfluoropropionic acid (PFPrA), 6:2 fluorotelomer alcohol (6:2 FTOH), 6:2 fluorotelomer sulfonic acid (6:2 FTSA), and 8:2 fluorotelomer sulfonic acid (8:2 FTSA)] for their capacity to activate nuclear receptors and to cause differential expression of genes involved in lipid metabolism. Cytotoxicity assays were run in parallel to exclude that observed differential gene expression was due to cytotoxicity. Based on the cytotoxicity assays and gene expression studies, PFOSA was shown to be more potent than other tested PFAS. PFOSA decreased the gene expression of crucial genes involved in bile acid synthesis and detoxification, cholesterol synthesis, bile acid and cholesterol transport, and lipid metabolism regulation. Except for 6:2 FTOH and 8:2 FTSA, all tested PFAS downregulated PPARA gene expression. The reporter gene assay also showed that 8:2 FTSA transactivated the farnesoid X receptor (FXR). Based on this study, PFOSA, 6:2 FTSA, and 8:2 FTSA were prioritized for further studies to confirm and understand their possible effects on hepatic lipid metabolism.

Recombinant Protein Production in Suspension Mammalian Cells Using the BacMam Baculovirus Expression System.

Mammalian cell lines are one of the best options when it comes to the production of complex proteins requiring specific glycosylation patterns. Plasmid DNA transfection and stable cell lines are frequently used for recombinant protein production, but they are expensive at large scale or can become time-consuming, respectively. The BacMam baculovirus (BV) is a safe and cost-effective platform to produce recombinant proteins in mammalian cells. The process of generating BacMam BVs is straightforward and similar to the generation of "insect" BVs, with different commercially available platforms. Although there are several protocols that describe recombinant protein expression with the BacMam BV in adherent cell lines, limited information is available on suspension cells. Therefore, it is of relevance to define the conditions to produce recombinant proteins in suspension cell cultures with BacMam BVs that facilitate bioprocess transfer to larger volumes. Here, we describe a method to generate a high titer BacMam BV stock and produce recombinant proteins in suspension HEK293 cells.

Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells.

The gene therapy field seeks cost-effective, large-scale production of recombinant adeno-associated virus (rAAV) vectors for high-dosage therapeutic applications. Although strategies like suspension cell culture and transfection optimization have shown moderate success, challenges persist for large-scale applications. To unravel molecular and cellular mechanisms influencing rAAV production, we conducted an SWATH-MS proteomic analysis of HEK293T cells transfected using standard, sub-optimal, and optimal conditions. Gene Ontology and pathway analysis revealed significant protein expression variations, particularly in processes related to cellular homeostasis, metabolic regulation, vesicular transport, ribosomal biogenesis, and cellular proliferation under optimal transfection conditions. This resulted in a 50% increase in rAAV titer compared with the standard protocol. Additionally, we identified modifications in host cell proteins crucial for AAV mRNA stability and gene translation, particularly regarding AAV capsid transcripts under optimal transfection conditions. Our study identified 124 host proteins associated with AAV replication and assembly, each exhibiting distinct expression pattern throughout rAAV production stages in optimal transfection condition. This investigation sheds light on the cellular mechanisms involved in rAAV production in HEK293T cells and proposes promising avenues for further enhancing rAAV titer during production.

Chemically Synthesized 1,2,3,4,6-Pentakis-O-Galloyl-ß-D-Glucopyranoside Blocks SARS-CoV-2 Spike Interaction with Host ACE-2 Receptor.

BACKGROUND: In the search for anti-COVID-19 therapy, 1,2,3,4,6-pentakis-O-galloyl-ßD-glucopyranoside, a natural polyphenolic compound isolated from many traditional medicinal herbs, has been reported as an RBD-ACE2 binding inhibitor and as a broad-spectrum anticoronaviral inhibitor targeting the main protease and RNA-dependent RNA polymerase of SARSCoV-2. To facilitate the structure-activity relationship studies of 1,2,3,4,6-pentakis-O-galloyl-ß-Dglucopyranoside, we describe its chemical synthesis and characterization, as well as its activity towards the SARS-CoV-2 spike interaction with host ACE2 receptor. METHODS: 1,2,3,4,6-Pentakis-O-galloyl-ß-D-glucopyranoside was synthesized in two quantitative steps from 3,4,5-tribenzyloxybenzoic acid and ß-D-glucopyranoside: DCC-mediated esterification and palladium-catalyzed per-debenzylation. The synthesized molecule was evaluated using a SARS-CoV-2 spike trimer (S1 + S2) ACE2 inhibitor screening colorimetric assay kit, SARS-CoV2 spike S1 RBD ACE2 inhibitor screening colorimetric assay kit, and a cellular neutralization assay using the Spike (SARS-CoV-2) Pseudotyped Lentivirus, ACE2-HEK293 recombinant cell line. RESULTS: The chemically synthesized product blocked the binding of the spike trimer of SARSCoV-2 to the human ACE2 receptor with IC50=22±2 µM. It also blocked ACE2:spike RBD binding with IC50=27±3 µM. Importantly, it inhibited the infectivity of SARS2-CoV2-Spike pseudotyped lentivirus on the ACE2 HEK293 cell line with IC50=20±2 µM. CONCLUSION: Overall, the chemically synthesized 1,2,3,4,6-pentakis-O-galloyl-ß-D-glucopyranoside represents a lead molecule to develop anti-SARS-CoV-2 therapies that block the initial stage of the viral infection by blocking the virus entry to the host cell.