Understanding the Manufacturing Process of Lipid Nanoparticles for mRNA Delivery Using Machine Learning.

Lipid nanoparticles (LNPs), used for mRNA vaccines against severe acute respiratory syndrome coronavirus 2, protect mRNA and deliver it into cells, making them an essential delivery technology for RNA medicine. The LNPs manufacturing process consists of two steps, the upstream process of preparing LNPs and the downstream process of removing ethyl alcohol (EtOH) and exchanging buffers. Generally, a microfluidic device is used in the upstream process, and a dialysis membrane is used in the downstream process. However, there are many parameters in the upstream and downstream processes, and it is difficult to determine the effects of variations in the manufacturing parameters on the quality of the LNPs and establish a manufacturing process to obtain high-quality LNPs. This study focused on manufacturing mRNA-LNPs using a microfluidic device. Extreme gradient boosting (XGBoost), which is a machine learning technique, identified EtOH concentration (flow rate ratio), buffer pH, and total flow rate as the process parameters that significantly affected the particle size and encapsulation efficiency. Based on these results, we derived the manufacturing conditions for different particle sizes (approximately 80 and 200 nm) of LNPs using Bayesian optimization. In addition, the particle size of the LNPs significantly affected the protein expression level of mRNA in cells. The findings of this study are expected to provide useful information that will enable the rapid and efficient development of mRNA-LNPs manufacturing processes using microfluidic devices.

Multitargeting strategy using lenvatinib and golvatinib: maximizing anti-angiogenesis activity in a preclinical cancer model.

Almost all cancers show intrinsic and/or evasive resistance to vascular endothelial growth factor (VEGF) inhibitors by multiple mechanisms. Serum angiopoietin-2 (Ang2) level has been proposed as a potential biomarker of VEGF inhibitor response in several cancers. From these clinical observations, the Ang2 and Tie2 (its receptor) axis has been focused on as a promising target. Here, we show a novel strategy to circumvent the resistance by combining multi-tyrosine kinase inhibitors lenvatinib (VEGF receptor, fibroblast growth factor receptor, and RET inhibitor) and golvatinib (E7050; c-Met, Tie2, and EphB4 inhibitor). Tie2 identifies a highly pro-angiogenic macrophage subset, Tie2-expressing macrophages (TEM). Angi-Tie2 and EphB4-EphrinB2 signaling plays critical roles in pericyte-mediated vessel stabilization. In vitro analyses suggested that golvatinib combined with lenvatinib inhibited pericyte-mediated vessel stabilization and TEM differentiation. In thyroid and endometrial cancer models, golvatinib and lenvatinib inhibited pericyte network development and TEM infiltration, resulting in severe perfusion disorder and massive apoptosis. Body weight loss was tolerable, and no macroscopic change was observed. These preclinical studies suggest that modulation of the tumor microenvironment by a strategic and well-tolerated combination of multi-targeting tyrosine kinase inhibitors may sensitize cancer to VEGF inhibitors.

Design and lyophilization of lipid nanoparticles for mRNA vaccine and its robust immune response in mice and nonhuman primates.

mRNA and lipid nanoparticles have emerged as powerful systems for the preparation of vaccines against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. The emergence of novel variants or the necessity of cold chain logistics for approved mRNA vaccines undermines the investigation of next-generation systems that could preserve both potency and stability. However, the correlation between lipid nanoparticle composition and activity is not fully explored. Here, we screened a panel of ionizable lipids in vivo and identified lead lipid nanoparticles with a branched-tail lipid structure. Buffer optimization allowed the determination of lyophilization conditions, where lipid nanoparticle-encapsulated mRNA encoding SARS-CoV-2 spike protein could induce robust immunogenicity in mice after 1 month of storage at 5°C and 25°C. Intramuscularly injected lipid nanoparticles distributed in conventional dendritic cells in mouse lymph nodes induced balanced T helper (Th) 1/Th2 responses against SARS-CoV-2 spike protein. In nonhuman primates, two doses of 10 or 100 µg of mRNA induced higher spike-specific binding geometric mean titers than those from a panel of SARS-CoV-2-convalescent human sera. Immunized sera broadly inhibited the viral entry receptor angiotensin-converting enzyme 2 (ACE2) from binding to the spike protein in all six strains tested, including variants of concern. These results could provide useful information for designing next-generation mRNA vaccines.

Peptidyl-prolyl-tRNA at the ribosomal P-site reacts poorly with puromycin.

Despite remarkable recent progress in our chemical and structural understanding of the mechanisms of peptide bond formation by the ribosome, only very limited information is available about whether amino acid side chains affect the rate of peptide bond formation. Here, we generated a series of peptidyl-tRNAs that end with different tRNA-attached amino acids in the P-site of the Escherichia coli ribosome and compared their reactivity with puromycin, a rapidly A-site-accessing analog of aminoacyl-tRNAs. Among the 20 amino acids examined, proline was found to receive exceptionally slow peptidyl transfer to puromycin. These results raise a possibility that the peptidyl transferase activity of the ribosome may have some specificity with regard to the P-site amino acids.

Genetically encoded but nonpolypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall.

The arrest sequence, FXXXXWIXXXXGIRAGP, of E. coli SecM interacts with the ribosomal exit tunnel, thereby interfering with translation elongation. Here, we studied this elongation arrest in vitro using purified translation components. While a simplest scenario would be that elongation is arrested beyond Pro166, the last arrest-essential amino acid, and that the Pro166 codon is positioned at the P site of the ribosomal peptidyl transferase center (PTC), our toeprint analyses revealed that the ribosome actually stalls when the Pro166 codon is positioned at the A site. Northern hybridization identification of the polypeptide bound tRNA and mass determination showed that the last amino acid of the arrested peptidyl-tRNA is Gly165, which is only inefficiently transferred to Pro166. Also, puromycin does not effectively release the arrested peptidyl-tRNA under the conditions of A site occupancy by Pro166-tRNA. These results reveal that secM-encoded Pro166-tRNA functions as a nonpolypeptide element in fulfilling SecM's role as a secretion monitor.