mRNA-encoded Cas13 treatment of Influenza via site-specific degradation of genomic RNA.

The CRISPR-Cas13 system has been proposed as an alternative treatment of viral infections. However, for this approach to be adopted as an antiviral, it must be optimized until levels of efficacy rival or exceed the performance of conventional approaches. To take steps toward this goal, we evaluated the influenza viral RNA degradation patterns resulting from the binding and enzymatic activity of mRNA-encoded LbuCas13a and two crRNAs from a prior study, targeting PB2 genomic and messenger RNA. We found that the genome targeting guide has the potential for significantly higher potency than originally detected, because degradation of the genomic RNA is not uniform across the PB2 segment, but it is augmented in proximity to the Cas13 binding site. The PB2 genome targeting guide exhibited high levels (>1 log) of RNA degradation when delivered 24 hours post-infection in vitro and maintained that level of degradation over time, with increasing multiplicity of infection (MOI), and across modern influenza H1N1 and H3N2 strains. Chemical modifications to guides with potent LbuCas13a function, resulted in nebulizer delivered efficacy (>1-2 log reduction in viral titer) in a hamster model of influenza (Influenza A/H1N1/California/04/09) infection given prophylactically or as a treatment (post-infection). Maximum efficacy was achieved with two doses, when administered both pre- and post-infection. This work provides evidence that mRNA-encoded Cas13a can effectively mitigate Influenza A infections opening the door to the development of a programmable approach to treating multiple respiratory infections.

In vitro experimental conditions and tools can influence the safety and biocompatibility results of antimicrobial electrospun biomaterials for wound healing.

Electrospun (ES) fibrous nanomaterials have been widely investigated as novel biomaterials. These biomaterials have to be safe and biocompatible; hence, they need to be tested for cytotoxicity before being administered to patients. The aim of this study was to develop a suitable and biorelevant in vitro cytotoxicity assay for ES biomaterials (e.g. wound dressings). We compared different in vitro cytotoxicity assays, and our model wound dressing was made from polycaprolactone and polyethylene oxide and contained chloramphenicol as the active pharmaceutical ingredient. Baby Hamster Kidney cells (BHK-21), human primary fibroblasts and MTS assays together with real-time cell analysis were selected. The extract exposure and direct contact safety evaluation setups were tested together with microscopic techniques. We found that while extract exposure assays are suitable for the initial testing, the biocompatibility of the biomaterial is revealed in in vitro direct contact assays where cell interactions with the ES wound dressing are evaluated. We observed significant differences in the experimental outcome, caused by the experimental set up modification such as cell line choice, cell medium and controls used, conducting the phosphate buffer washing step or not. A more detailed technical protocol for the in vitro cytotoxicity assessment of ES wound dressings was developed.

Immune reactivity of two biological models to vaccination with inactivated vaccine QazVac against coronavirus infection COVID-19.

INTRODUCTION: Specific prevention of a number of infectious diseases has been introduced into the vaccination schedule. The production of immunoprophylactic drugs, in order to establish standard properties, including safety and specific effectiveness, requires strict adherence to manufacturing regulations, and the reliability of the results obtained requires monitoring of these parameters. The specific effectiveness of vaccine preparations is standardized according to the indicators of stimulation of specific antibody response formed in the body of vaccinated model biological objects. OBJECTIVE: Determination of the immune reactivity of white mice to vaccination with the QazVac vaccine to establish the possibility of using them as a biological model in assessing the immunogenicity of the vaccine instead of Syrian hamsters. MATERIALS AND METHODS: The immune reactivity of model animals was assessed by the seroconversion rate, dynamics of antibody titers to the SARS-CoV-2 virus formed in the body after vaccination with the test vaccine. In the case of seropositivity of animals before administration of vaccine or placebo, the level of immune reactivity was calculated by the difference in antibody titers between control and vaccinated animals or by the difference in antibody titers before and after immunization. Specific antibodies were detected and their titer was determined using a neutralization reaction. RESULTS: The research results showed that the tested biological models had approximately the same immune reactivity to the administration of the QazVac vaccine, confirmed by the level and dynamics of antibody titers. When analyzing the fold increase in antibody titers in comparison to those of control animals, Syrian hamsters were more reactive compared to mice. But SPF white mice were standardized in their lack of the immune reactivity to SARS-CoV-2 virus before the immunization. CONCLUSION: The data obtained indicate that the immune reactivity of white mice to the administration of the QazVac vaccine in terms of the rate and dynamics of the formation of virus-neutralizing antibodies is approximately equivalent to the immune reactivity of Syrian hamsters. Before immunization with the vaccine, SPF white mice, in contrast to Syrian hamsters, do not have humoral immunity specific to the SARS-CoV-2 virus. The immune reactivity equivalent to that observed of Syrian hamsters and the absence of antibodies to the SARS-CoV-2 virus at a baseline indicate the superiority of the use of white mice in assessing the immunogenicity of vaccines against COVID-19 and/or obtaining specific factors of humoral immunity.

Association between Ultradian Rhythms of Body Temperature in Small Mammals and Earth's Crust Stress.

Association was assessed between the data harvested by a long-baseline laser interference deformograph and the dynamics of body temperature (BT) in hamsters deprived of natural daily light-darkness changes. The power spectral data revealed the positive correlation between simultaneous time series of hamster BT and the Earth's crust deformation (ECD). The superposed epoch analysis established an association between abrupt upstrokes of hamster BT and ECD increments. Thus, the direct relationships between BT dynamics (reflecting predominance of sympathetic part of autonomic nervous system) and ECD (according to long-baseline laser interference deformography) were established. The study observed synchronization of the free-running circadian rhythm of hamster BT with the tidal stress in Earth's lithosphere. Further studies are needed to find the physical factor underlying the revealed relationships.

SARS-CoV-2 envelope protein-derived extracellular vesicles act as potential media for viral spillover.

Extracellular vesicles (EVs) are shown to be a novel viral transmission model capable of increasing a virus's tropism. According to our earlier research, cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or transfected with envelope protein plasmids generate a novel type of EVs that are micrometer-sized and able to encase virus particles. Here, we showed the capacity of these EVs to invade various animals both in vitro and in vivo independent of the angiotensin-converting enzyme 2 receptor. First, via macropinocytosis, intact EVs produced from Vero E6 (monkey) cells were able to enter cells from a variety of animals, including cats, dogs, bats, hamsters, and minks, and vice versa. Second, when given to zebrafish with cutaneous wounds, the EVs showed favorable stability in aqueous environments and entered the fish. Moreover, infection of wild-type (WT) mice with heterogeneous EVs carrying SARS-CoV-2 particles led to a strong cytokine response and a notable amount of lung damage. Conversely, free viral particles did not infect WT mice. These results highlight the variety of processes behind viral transmission and cross-species evolution by indicating that EVs may be possible vehicles for SARS-CoV-2 spillover and raising risk concerns over EVs' potential for viral gene transfer.

An intranasal nanoparticle STING agonist protects against respiratory viruses in animal models.

Respiratory viral infections cause morbidity and mortality worldwide. Despite the success of vaccines, vaccination efficacy is weakened by the rapid emergence of viral variants with immunoevasive properties. The development of an off-the-shelf, effective, and safe therapy against respiratory viral infections is thus desirable. Here, we develop NanoSTING, a nanoparticle formulation of the endogenous STING agonist, 2'-3' cGAMP, to function as an immune activator and demonstrate its safety in mice and rats. A single intranasal dose of NanoSTING protects against pathogenic strains of SARS-CoV-2 (alpha and delta VOC) in hamsters. In transmission experiments, NanoSTING reduces the transmission of SARS-CoV-2 Omicron VOC to naïve hamsters. NanoSTING also protects against oseltamivir-sensitive and oseltamivir-resistant strains of influenza in mice. Mechanistically, NanoSTING upregulates locoregional interferon-dependent and interferon-independent pathways in mice, hamsters, as well as non-human primates. Our results thus implicate NanoSTING as a broad-spectrum immune activator for controlling respiratory virus infection.

Three SARS-CoV-2 spike protein variants delivered intranasally by measles and mumps vaccines are broadly protective.

As the new SARS-CoV-2 Omicron variants and subvariants emerge, there is an urgency to develop intranasal, broadly protective vaccines. Here, we developed highly efficacious, intranasal trivalent SARS-CoV-2 vaccine candidates (TVC) based on three components of the MMR vaccine: measles virus (MeV), mumps virus (MuV) Jeryl Lynn (JL1) strain, and MuV JL2 strain. Specifically, MeV, MuV-JL1, and MuV-JL2 vaccine strains, each expressing prefusion spike (preS-6P) from a different variant of concern (VoC), were combined to generate TVCs. Intranasal immunization of IFNAR1-/- mice and female hamsters with TVCs generated high levels of S-specific serum IgG antibodies, broad neutralizing antibodies, and mucosal IgA antibodies as well as tissue-resident memory T cells in the lungs. The immunized female hamsters were protected from challenge with SARS-CoV-2 original WA1, B.1.617.2, and B.1.1.529 strains. The preexisting MeV and MuV immunity does not significantly interfere with the efficacy of TVC. Thus, the trivalent platform is a promising next-generation SARS-CoV-2 vaccine candidate.

Advances in serum-free media for CHO cells: From traditional serum substitutes to microbial-derived substances.

The Chinese hamster ovary (CHO) cell is an epithelial-like cell that produces proteins with post-translational modifications similar to human glycosylation. It is widely used in the production of recombinant therapeutic proteins and monoclonal antibodies. Culturing CHO cells typically requires the addition of a certain proportion of fetal bovine serum (FBS) to maintain cell proliferation and passaging. However, serum is characterized by its complex composition, batch-to-batch variability, high cost, and potential risk of exogenous contaminants such as mycoplasma and viruses, which impact the purity and safety of the synthesized proteins. Therefore, search for serum alternatives and development of serum-free media for CHO-based protein biomanufacturing are of great significance. This review systematically summarizes the application advantages of CHO cells and strategies for high-density expression. It highlights the developmental trends of serum substitutes from human platelet lysates to animal-free extracts and microbial-derived substances and elucidates the mechanisms by which these substitutes enhance CHO cell culture performance and recombinant protein production, aiming to provide theoretical guidance for exploring novel serum alternatives and developing serum-free media for CHO cells.

Nanoscale intracellular ultrastructures affected by osmotic pressure using small-angle X-ray scattering.

Although intracellular ultrastructures have typically been studied using microscopic techniques, it is difficult to observe ultrastructures at the submicron scale of living cells due to spatial resolution (fluorescence microscopy) or high vacuum environment (electron microscopy). We investigate the nanometer scale intracellular ultrastructures of living CHO cells in various osmolality using small-angle X-ray scattering (SAXS), and especially the structures of ribosomes, DNA double helix, and plasma membranes in-cell environment are observed. Ribosomes expand and contract in response to osmotic pressure, and the inter-ribosomal correlation occurs under isotonic and hyperosmolality. The DNA double helix is not dependent on the osmotic pressure. Under high osmotic pressure, the plasma membrane folds into form a multilamellar structure with a periodic length of about 6 nm. We also study the ultrastructural changes caused by formaldehyde fixation, freezing and heating.

Development of a live attenuated vaccine candidate for equid alphaherpesvirus 1 control: a step towards efficient protection.

Equid alphaherpesvirus 1 (EqAHV1) is a viral pathogen known to cause respiratory disease, neurologic syndromes, and abortion storms in horses. Currently, there are no vaccines that provide complete protection against EqAHV1. Marker vaccines and the differentiation of infected and vaccinated animals (DIVA) strategy are effective for preventing and controlling outbreaks but have not been used for the prevention of EqAHV1 infection. Glycoprotein 2 (gp2), located on the envelope of viruses (EqAHV1), exhibits high antigenicity and functions as a molecular marker for DIVA. In this study, a series of EqAHV1 mutants with deletion of gp2 along with other virulence genes (TK, UL24/TK, gI/gE) were engineered. The mutant viruses were studied in vitro and then in an in vivo experiment using Golden Syrian hamsters to assess the extent of viral attenuation and the immune response elicited by the mutant viruses in comparison to the wild-type (WT) virus. Compared with the WT strain, the YM2019 Δgp2, ΔTK/gp2, and ΔUL24/TK/gp2 strains exhibited reduced growth in RK-13 cells, while the ΔgI/gE/gp2 strain exhibited significantly impaired proliferation. The YM2019 Δgp2 strain induced clinical signs and mortality in hamsters. In contrast, the YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 variants displayed diminished pathogenicity, causing no observable clinical signs or fatalities. Immunization with nasal vaccines containing YM2019 ΔTK/gp2 and ΔUL24/TK/gp2 elicited a robust immune response in hamsters. In particular, compared with the vaccine containing the ΔTK/gp2 strain, the vaccine containing the ΔUL24/TK/gp2 strain demonstrated enhanced immune protection upon challenge with the WT virus. Furthermore, an ELISA for gp2 was established and refined to accurately differentiate between infected and vaccinated animals. These results confirm that the ΔUL24/TK/gp2 strain is a safe and effective live attenuated vaccine candidate for controlling EqAHV1 infection.

The state of play of rodent models for the study of Clostridioides difficile infection.

Clostridioides difficile is the most common cause of nosocomial antibiotic-associated diarrhoea and is responsible for a spectrum of diseases characterized by high levels of recurrence and morbidity. In some cases, complications can lead to death. Currently, several types of animal models have been developed to study various aspects of C. difficile infection (CDI), such as colonization, virulence, transmission and recurrence. These models have also been used to test the role of environmental conditions, such as diet, age and microbiome that modulate infection outcome, and to evaluate several therapeutic strategies. Different rodent models have been used successfully, such as the hamster model and the gnotobiotic and conventional mouse models. These models can be applied to study either the initial CDI infectious process or recurrences. The applications of existing rodent models and their advantages and disadvantages are discussed here.

A highly effective ferritin-based divalent nanoparticle vaccine shields Syrian hamsters against lethal Nipah virus.

The Nipah virus (NiV), a highly deadly bat-borne paramyxovirus, poses a substantial threat due to recurrent outbreaks in specific regions, causing severe respiratory and neurological diseases with high morbidity. Two distinct strains, NiV-Malaysia (NiV-M) and NiV-Bangladesh (NiV-B), contribute to outbreaks in different geographical areas. Currently, there are no commercially licensed vaccines or drugs available for prevention or treatment. In response to this urgent need for protection against NiV and related henipaviruses infections, we developed a novel homotypic virus-like nanoparticle (VLP) vaccine co-displaying NiV attachment glycoproteins (G) from both strains, utilizing the self-assembling properties of ferritin protein. In comparison to the NiV G subunit vaccine, our nanoparticle vaccine elicited significantly higher levels of neutralizing antibodies and provided complete protection against a lethal challenge with NiV infection in Syrian hamsters. Remarkably, the nanoparticle vaccine stimulated the production of antibodies that exhibited superior cross-reactivity to homologous or heterologous henipavirus. These findings underscore the potential utility of ferritin-based nanoparticle vaccines in providing both broad-spectrum and long-term protection against NiV and emerging zoonotic henipaviruses challenges.

Haloketones: A class of unregulated priority DBPs with high contribution to drinking water cytotoxicity.

Although unregulated aliphatic disinfection byproducts (DBPs) had a much higher concentration and cytotoxicity than known aromatic DBPs, a recent study indicated that seven classes of regulated and unregulated priority DBPs (one and two-carbon-atom DBPs) just accounted for 16.2% of disinfected water cytotoxicity in the U.S., meaning some of the highly toxic aliphatic DBPs may be overlooked. Haloketones (HKs) are an essential class of priority DBPs with a 1-100 µg/L concentration in drinking water but lack cytotoxicity data. This study investigated the cytotoxicity of seven HKs using Chinese hamster ovary (CHO) cells. The order for cytotoxicity of HKs from most to least toxic was: 1,3-dichloroacetone (LC50: 1.0 ± 0.20 µM) ≈ 1,3-dibromoacetone (1.5 ± 0.19 µM) ≈ bromoacetone (1.9 ± 0.49 µM) > chloroacetone (4.3 ± 0.22 µM) > 1,1,3-trichloropropanone (6.6 ± 0.46 µM) > 1,1,1-trichloroacetone (222 ± 7.7 µM) > hexachloroacetone (3269 ± 344 µM). The cytotoxicity of HKs was higher than most regulated and priority aliphatic DBPs in mono-halogenated, di-halogenated, and tri-halogenated categories. A prediction model of HK cytotoxicity was developed based on the quantitative structure-activity relationship (QSAR), optimizing structures and computing descriptors with Gaussian 09 W. The average concentrations of HKs in representative drinking water samples from South Carolina (U.S.) and Suzhou (China) were 12.4 and 0.9 µg/L, respectively, accounting for 18.8% and 1.7% of their specific total DBPs measured (i.e. not TOX). For South Carolina drinking water, their contributions to total calculated additive cytotoxicity of aliphatic DBPs and overall drinking water cytotoxicity were 86.7% and 14.0%, respectively, demonstrating that HKs are an essential class of overlooked DBPs with a high contribution to drinking water cytotoxicity. Our study can help to explain the conflict that why regulated and priority DBPs (except HKs) just accounted for 16% of chlorinated drinking water cytotoxicity even enough they had much higher concentration and cytotoxicity than known aromatic DBPs.

Protocol for microfluidic single-cell cultivation and live-cell imaging of Chinese hamster ovary suspension cell lines.

Microfluidic single-cell cultivation (MSCC) is a powerful tool for investigating the cellular behavior of various cell types at the single-cell level. Here, we present a protocol specifically developed for the reliable and reproducible MSCC of industrially relevant Chinese hamster ovary (CHO) suspension cell lines. We summarize critical experimental steps from the initial seed train up to the final MSCC experiment, with a special focus on pre-culture management and medium preparation, device inoculation, and the establishment of a constant medium perfusion.

AZD3152 neutralizes SARS-CoV-2 historical and contemporary variants and is protective in hamsters and well tolerated in adults.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in variants that can escape neutralization by therapeutic antibodies. Here, we describe AZD3152, a SARS-CoV-2-neutralizing monoclonal antibody designed to provide improved potency and coverage against emerging variants. AZD3152 binds to the back left shoulder of the SARS-CoV-2 spike protein receptor binding domain and prevents interaction with the human angiotensin-converting enzyme 2 receptor. AZD3152 potently neutralized a broad panel of pseudovirus variants, including the currently dominant Omicron variant JN.1 but has reduced potency against XBB subvariants containing F456L. In vitro studies confirmed F456L resistance and additionally identified T415I and K458E as escape mutations. In a Syrian hamster challenge model, prophylactic administration of AZD3152 protected hamsters from weight loss and inflammation-related lung pathologies and reduced lung viral load. In the phase 1 sentinel safety cohort of the ongoing SUPERNOVA study (ClinicalTrials.gov: NCT05648110), a single 600-mg intramuscular injection of AZD5156 (containing 300 mg each of AZD3152 and cilgavimab) was well tolerated in adults through day 91. Observed serum concentrations of AZD3152 through day 91 were similar to those observed with cilgavimab and consistent with predictions for AZD7442, a SARS-CoV-2-neutralizing antibody combination of cilgavimab and tixagevimab, in a population pharmacokinetic model. On the basis of its pharmacokinetic characteristics, AZD3152 is predicted to provide durable protection against symptomatic coronavirus disease 2019 caused by susceptible SARS-CoV-2 variants, such as JN.1, in humans.

Manufacturing and preclinical toxicity of GLP grade gene deleted attenuated Leishmania donovani parasite vaccine.

Centrin1 gene deleted Leishmania donovani parasite (LdCen1-/-) was developed and extensively tested experimentally as an intracellular stage-specific attenuated and immunoprotective live parasite vaccine candidate ex vivo using human PBMCs and in vivo in animals. Here we report manufacturing and pre-clinical evaluation of current Good-Laboratory Practice (cGLP) grade LdCen1-/- parasites, as a prerequisite before proceeding with clinical trials. We screened three batches of LdCen1-/- parasites manufactured in bioreactors under cGLP conditions, for their consistency in genetic stability, attenuation, and safety. One such batch was preclinically tested using human PBMCs and animals (hamsters and dogs) for its safety and protective immunogenicity. The immunogenicity of the CGLP grade LdCen1-/- parasites was similar to one grown under laboratory conditions. The cGLP grade LdCen1-/- parasites were found to be safe and non-toxic in hamsters and dogs even at 3 times the anticipated vaccine dose. When PBMCs from healed visceral leishmaniasis (VL) cases were infected with cGLP LdCen1-/-, there was a significant increase in the stimulation of cytokines that contribute to protective responses against VL. This effect, measured by multiplex ELISA, was greater than that observed in PBMCs from healthy individuals. These results suggest that cGLP grade LdCen1-/- manufactured under cGMP complaint conditions can be suitable for future clinical trials.

Characterization of a natural 'dead-end' variant of Schmallenberg virus.

Schmallenberg virus (SBV) belongs to the Simbu serogroup within the family Peribunyaviridae, genus Orthobunyavirus and is transmitted by Culicoides biting midges. Infection of naïve ruminants in a critical phase of gestation may lead to severe congenital malformations. Sequence analysis from viremic animals revealed a very high genome stability. In contrast, sequence variations are frequently described for SBV from malformed fetuses. In addition to S segment mutations, especially within the M segment encoding the major immunogen Gc, point mutations or genomic deletions are also observed. Analysis of the SBV_D281/12 isolate from a malformed fetus revealed multiple point mutations in all three genome segments. It also has a large genomic deletion in the antigenic domain encoded by the M segment compared to the original SBV reference strain 'BH80/11' isolated from viremic blood in 2011. Interestingly, SBV_D281/12 showed a marked replication deficiency in vitro in Culicoides sonorensis cells (KC cells), but not in standard baby hamster kidney cells (BHK-21). We therefore generated a set of chimeric viruses of rSBV_D281/12 and wild-type rSBV_BH80/11 by reverse genetics, which were characterized in both KC and BHK-21 cells. It could be shown that the S segment of SBV_D281/12 is responsible for the replication deficit and that it acts independently from the large deletion within Gc. In addition, a single point mutation at position 111 (S to N) of the nucleoprotein was identified as the critical mutation. Our results suggest that virus variants found in malformed fetuses and carrying characteristic genomic mutations may have a clear 'loss of fitness' for their insect hosts in vitro. It can also be concluded that such mutations lead to virus variants that are no longer part of the natural transmission cycle between mammalian and insect hosts. Interestingly, analysis of a series of SBV sequences confirmed the S111N mutation exclusively in samples of malformed fetuses and not in blood from viremic animals. The characterization of these changes will allow the definition of protein functions that are critical for only one group of hosts.

Hamster spermatozoa incorporate hypotaurine via TauT for self-protection.

In brief: Mammalian spermatozoa actively generate reactive oxygen species (ROS) during capacitation, a maturational process necessary for fertilization in vivo. This study shows that hypotaurine, a precursor of taurine present in the oviduct, is incorporated and concentrated in hamster sperm cells via the taurine transporter, TauT, for cytoprotection against self-produced ROS. Abstract: To achieve fertilization competence, mammalian spermatozoa undergo capacitation, during which they actively generate reactive oxygen species (ROS). Therefore, mammalian spermatozoa must protect themselves from these self-generated ROS. The mammalian oviductal fluid is rich in hypotaurine, a taurine precursor, which reportedly protects mammalian spermatozoa, including those of hamsters, from ROS; however, its precise mechanism remains unknown. This study aimed to elucidate the mechanism underlying hypotaurine-mediated protection of spermatozoa from ROS using hamsters, particularly focusing on the taurine/hypotaurine transporter TauT. The effect of hypotaurine on sperm motility and ROS levels was tested using sperm motility analysis and the CellROX dye and luminol assays. RNA sequencing analysis was performed to verify TauT expression. We found that hypotaurine was necessary for maintaining sperm motility and hyperactivated motility. Hypotaurine did not scavenge extracellular ROS but lowered intracellular ROS levels and was incorporated and concentrated in hamster spermatozoa. TauT was detected at both mRNA and protein levels. ß-Alanine blocked hypotaurine transport, increased intracellular ROS levels, and inhibited hyperactivation. Elimination of Na+ or Cl- ions inhibited hypotaurine transport and increased intracellular ROS levels. Thus, these results indicated that hamster spermatozoa incorporated and concentrated hypotaurine in sperm cells via TauT to protect themselves from self-generated ROS.

Metabolic engineering of CHO cells towards cysteine prototrophy and systems analysis of the ensuing phenotype.

Chinese hamster ovary (CHO) cells require cysteine for growth and productivity in fed-batch cultures. In intensified processes, supplementation of cysteine at high concentrations is a challenge due to its limited solubility and instability in solution. Methionine can be converted to cysteine (CYS) but key enzymes, cystathionine beta-synthase (Cbs) and cystathionine gamma-lyase (Cth), are not active in CHO cells resulting in accumulation of an intermediate, homocysteine (HCY), in cell culture milieu. In this study, Cbs and Cth were overexpressed in CHO cells to confer cysteine prototrophy, i.e., the ability to grow in a cysteine free environment. These pools (CbCt) needed homocysteine and beta-mercaptoethanol (ßME) to grow in CYS-free medium. To increase intracellular homocysteine levels, Gnmt was overexpressed in CbCt pools. The resultant cell pools (GnCbCt), post adaptation in CYS-free medium with decreasing residual HCY and ßME levels, were able to proliferate in the HCY-free, ßME-free and CYS-free environment. Interestingly, CbCt pools were also able to be adapted to grow in HCY-free and CYS-free conditions, albeit at significantly higher doubling times than GnCbCt cells, but couldn't completely adapt to ßME-free conditions. Further, single cell clones derived from the GnCbCt cell pool had a wide range in expression levels of Cbs, Cth and Gnmt and, when cultivated in CYS-free fed-batch conditions, performed similarly to the wild type (WT) cell line cultivated in CYS supplemented fed-batch culture. Intracellular metabolomic analysis showed that HCY and glutathione (GSH) levels were lower in the CbCt pool in CYS-free conditions but were restored closer to WT levels in the GnCbCt cells cultivated in CYS-free conditions. Transcriptomic analysis showed that GnCbCt cells upregulated several genes encoding transporters as well as methionine catabolism and transsulfuration pathway enzymes that support these cells to biosynthesize cysteine effectively. Further, 'omics analysis suggested CbCt pool was under ferroptotic stress in CYS-free conditions, which, when inhibited, enhanced the growth and viability of these cells in CYS-free conditions.

Increased stable integration efficiency in CHO cells through enhanced nuclear localization of Bxb1 serine integrase.

BACKGROUND: Mammalian display is an appealing technology for therapeutic antibody development. Despite the advantages of mammalian display, such as full-length IgG display with mammalian glycosylation and its inherent ability to select antibodies with good biophysical properties, the restricted library size and large culture volumes remain challenges. Bxb1 serine integrase is commonly used for the stable genomic integration of antibody genes into mammalian cells, but presently lacks the efficiency required for the display of large mammalian display libraries. To increase the Bxb1 integrase-mediated stable integration efficiency, our study investigates factors that potentially affect the nuclear localization of Bxb1 integrase. METHODS: In an attempt to enhance Bxb1 serine integrase-mediated integration efficiency, we fused various nuclear localization signals (NLS) to the N- and C-termini of the integrase. Concurrently, we co-expressed multiple proteins associated with nuclear transport to assess their impact on the stable integration efficiency of green fluorescent protein (GFP)-encoding DNA and an antibody display cassette into the genome of Chinese hamster ovary (CHO) cells containing a landing pad for Bxb1 integrase-mediated integration. RESULTS: The nucleoplasmin NLS from Xenopus laevis, when fused to the C-terminus of Bxb1 integrase, demonstrated the highest enhancement in stable integration efficiency among the tested NLS fusions, exhibiting over a 6-fold improvement compared to Bxb1 integrase lacking an NLS fusion. Subsequent additions of extra NLS fusions to the Bxb1 integrase revealed an additional 131% enhancement in stable integration efficiency with the inclusion of two copies of C-terminal nucleoplasmin NLS fusions. Further improvement was achieved by co-expressing the Ran GTPase-activating protein (RanGAP). Finally, to validate the applicability of these findings to more complex proteins, the DNA encoding the membrane-bound clinical antibody abrilumab was stably integrated into the genome of CHO cells using Bxb1 integrase with two copies of C-terminal nucleoplasmin NLS fusions and co-expression of RanGAP. This approach demonstrated over 14-fold increase in integration efficiency compared to Bxb1 integrase lacking an NLS fusion. CONCLUSIONS: This study demonstrates that optimizing the NLS sequence fusion for Bxb1 integrase significantly enhances the stable genomic integration efficiency. These findings provide a practical approach for constructing larger libraries in mammalian cells through the stable integration of genes into a genomic landing pad.