'Omics driven discoveries of gene targets for apoptosis attenuation in CHO cells.

Chinese hamster ovary (CHO) cells are widely used in biopharmaceutical production. Improvements to cell lines and bioprocesses are constantly being explored. One of the major limitations of CHO cell culture is that the cells undergo apoptosis, leading to rapid cell death, which impedes reaching high recombinant protein titres. While several genetic engineering strategies have been successfully employed to reduce apoptosis, there is still room to further enhance CHO cell lines performance. 'Omics analysis is a powerful tool to better understand different phenotypes and for the identification of gene targets for engineering. Here, we present a comprehensive review of previous CHO 'omics studies that revealed changes in the expression of apoptosis-related genes. We highlight targets for genetic engineering that have reduced, or have the potential to reduce, apoptosis or to increase cell proliferation in CHO cells, with the final aim of increasing productivity.

Attenuating apoptosis in Chinese hamster ovary cells for improved biopharmaceutical production.

Chinese hamster ovary (CHO) cells are the predominant host cell line for the production of biopharmaceuticals, a growing industry currently worth more than $188 billion USD in global sales. CHO cells undergo programmed cell death (apoptosis) following different stresses encountered in cell culture, such as substrate limitation, accumulation of toxic by-products, and mechanical shear, hindering production. Genetic engineering strategies to reduce apoptosis in CHO cells have been investigated with mixed results. In this review, a contemporary understanding of the real complexity of apoptotic mechanisms and signaling pathways is described; followed by an overview of antiapoptotic cell line engineering strategies tested so far in CHO cells.

Multifunctional nanoworms and nanorods through a one-step aqueous dispersion polymerization.

Producing synthetic soft worm and rod structures with multiple chemical functionalities on the surface would provide potential utility in drug delivery, nanoreactors, tissue engineering, diagnostics, rheology modifiers, enzyme mimics, and many other applications. Here, we have synthesized multifunctional worms and rods directly in water using a one-step reversible addition-fragmentation chain transfer (RAFT)-mediated dispersion polymerization at high weight fractions of polymer (>10 wt %). The chain-end functionalities included alkyne, pyridyl disulfide, dopamine, ß-thiolactone, and biotin groups. These groups could further be converted or coupled with biomolecules or polymers. We further demonstrated a nanorod colorimetric system with good control over the attachment of fluorescent probes.

Intracellular trafficking pathways for nuclear delivery of plasmid DNA complexed with highly efficient endosome escape polymers.

Understanding the pathways for nuclear entry could see vast improvements in polymer design for the delivery of genetic materials to cells. Here, we use a novel diblock copolymer complexed with plasmid DNA (pDNA) to determine both its cellular entry and nuclear pathways. The diblock copolymer (A-C3) is specifically designed to bind and protect pDNA, release it at a specific time, but more importantly, rapidly escape the endosome. The copolymer was taken up by HEK293 cells preferentially via the clathrin-mediated endocytosis (CME) pathway, and the pDNA entered the nucleus to produce high gene expression levels in all cells after 48 h, a similar observation to the commercially available polymer transfection agent, PEI Max. This demonstrates that the polymers must first escape the endosome and then mediate transport of pDNA to the nucleus for occurrence of gene expression. The amount of pDNA within the nucleus was found to be higher for our A-C3 polymer than PEI Max, with our polymer delivering 7 times more pDNA than PEI Max after 24 h. We further found that entry into the nucleus was primarily through the small nuclear pores and did not occur during mitosis when the nuclear envelope becomes compromised. The observation that the polymers are also found in the nucleus supports the hypothesis that the large pDNA/polymer complex (size ~200 nm) must dissociate prior to nucleus entry and that cationic and hydrophobic monomer units on the polymer may facilitate active transport of the pDNA through the nuclear pore.

Broad immunity to SARS-CoV-2 variants of concern mediated by a SARS-CoV-2 receptor-binding domain protein vaccine.

BACKGROUND: The SARS-CoV-2 global pandemic has fuelled the generation of vaccines at an unprecedented pace and scale. However, many challenges remain, including: the emergence of vaccine-resistant mutant viruses, vaccine stability during storage and transport, waning vaccine-induced immunity, and concerns about infrequent adverse events associated with existing vaccines. METHODS: We report on a protein subunit vaccine comprising the receptor-binding domain (RBD) of the ancestral SARS-CoV-2 spike protein, dimerised with an immunoglobulin IgG1 Fc domain. These were tested in conjunction with three different adjuvants: a TLR2 agonist R4-Pam2Cys, an NKT cell agonist glycolipid α-Galactosylceramide, or MF59® squalene oil-in-water adjuvant, using mice, rats and hamsters. We also developed an RBD-human IgG1 Fc vaccine with an RBD sequence of the immuno-evasive beta variant (N501Y, E484K, K417N). These vaccines were also tested as a heterologous third dose booster in mice, following priming with whole spike vaccine. FINDINGS: Each formulation of the RBD-Fc vaccines drove strong neutralising antibody (nAb) responses and provided durable and highly protective immunity against lower and upper airway infection in mouse models of COVID-19. The 'beta variant' RBD vaccine, combined with MF59® adjuvant, induced strong protection in mice against the beta strain as well as the ancestral strain. Furthermore, when used as a heterologous third dose booster, the RBD-Fc vaccines combined with MF59® increased titres of nAb against other variants including alpha, delta, delta+, gamma, lambda, mu, and omicron BA.1, BA.2 and BA.5. INTERPRETATION: These results demonstrated that an RBD-Fc protein subunit/MF59® adjuvanted vaccine can induce high levels of broadly reactive nAbs, including when used as a booster following prior immunisation of mice with whole ancestral-strain spike vaccines. This vaccine platform offers a potential approach to augment some of the currently approved vaccines in the face of emerging variants of concern, and it has now entered a phase I clinical trial. FUNDING: This work was supported by grants from the Medical Research Future Fund (MRFF) (2005846), The Jack Ma Foundation, National Health and Medical Research Council of Australia (NHMRC; 1113293) and Singapore National Medical Research Council (MOH-COVID19RF-003). Individual researchers were supported by an NHMRC Senior Principal Research Fellowship (1117766), NHMRC Investigator Awards (2008913 and 1173871), Australian Research Council Discovery Early Career Research Award (ARC DECRA; DE210100705) and philanthropic awards from IFM investors and the A2 Milk Company.

Preclinical development of a molecular clamp-stabilised subunit vaccine for severe acute respiratory syndrome coronavirus 2.

OBJECTIVES: Efforts to develop and deploy effective vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continue at pace. Here, we describe rational antigen design through to manufacturability and vaccine efficacy of a prefusion-stabilised spike (S) protein, Sclamp, in combination with the licensed adjuvant MF59 'MF59C.1' (Seqirus, Parkville, Australia). METHODS: A panel recombinant Sclamp proteins were produced in Chinese hamster ovary and screened in vitro to select a lead vaccine candidate. The structure of this antigen was determined by cryo-electron microscopy and assessed in mouse immunogenicity studies, hamster challenge studies and safety and toxicology studies in rat. RESULTS: In mice, the Sclamp vaccine elicits high levels of neutralising antibodies, as well as broadly reactive and polyfunctional S-specific CD4+ and cytotoxic CD8+ T cells in vivo. In the Syrian hamster challenge model (n = 70), vaccination results in reduced viral load within the lung, protection from pulmonary disease and decreased viral shedding in daily throat swabs which correlated strongly with the neutralising antibody level. CONCLUSION: The SARS-CoV-2 Sclamp vaccine candidate is compatible with large-scale commercial manufacture, stable at 2-8°C. When formulated with MF59 adjuvant, it elicits neutralising antibodies and T-cell responses and provides protection in animal challenge models.

Bisphosphonate Stabilized Calcium Phosphate Nanoparticles for Effective Delivery of Plasmid DNA to Macrophages.

Calcium phosphate (CP) nanoparticles (NPs) have been used in various applications for delivery of nucleic acid (NA) cargos with ideal biocompatibility and safety. However, some critical issues such as poor stability and aggregation in water solution hinder the industrial application of CP NPs. To further utilize CP NPs for NA delivery, this study specifically focused on the modification of CP NPs to achieve a rapid synthesis and improvement on dispersibility and colloidal stability by using a bisphosphonate (BP) and BSA (named as BCP NPs). Compared with CP NPs, BCP NPs show better stability and dispersity in the cell culture medium, higher efficiency in cellular uptake, and faster dissolution in acidic environments, which are essential requirements for NA vaccine delivery. The cell viability (MTT) assay indicates that BCP NPs have a similar or lower cytotoxicity than free alendronate and Lipofectamine 2000 reagent (L2K) to macrophages (MΦs), a type of typical antigen-presenting cells (APCs). Furthermore, BCP NPs exhibited 85% plasmid DNA (pDNA) loading efficiency and a good endosome escape property. Using a plasmid expressing enhanced green fluorescent protein (pEGFP) as a model system, we showed that BCP NP transfection resulted in a high-level EGFP expression in MΦs, which was even greater than the commercial L2K and electroporation method. This is the first application of a low amount of BP and BSA to modify CP-based NPs with low MΦ cytotoxicity and favorable dispersity, and our data suggest these BCP NPs are an excellent delivery platform for pDNA to MΦs.

Media composition modulates human embryonic stem cell morphology and may influence preferential lineage differentiation potential.

Undifferentiated human embryonic stem cells have a distinct morphology (hESC). Changes in cell morphology during culture can be indicative of differentiation. hESC, maintained in diverse medias, demonstrated alterations in morphological parameters and subsequent alterations in underlying transcript expression and lineage differentiation. Analysis of morphological parameters showed distinct and significant differences between the undefined, less defined and Xeno-free medias while still maintaining pluripotency markers. This suggested that the less defined media may be creating dynamic instability in the cytoskeleton, with the cytoskeleton becoming more stabilised in the Xeno-free media as demonstrated by smaller and rounder cells. Examination of early lineage markers during undirected differentiation using d5 embryoid bodies demonstrated increased mesodermal lineage preference as compared to endodermal or ectoderm in cells originally cultured in Xeno-free media. Undefined media showed preference for mesoderm and ectoderm lineages, while less defined media (BSA present) demonstrated no preference. These data reveal that culture media may produce fundamental changes in cell morphology which are reflected in early lineage differentiation choice.