Achieving precise control of nanoparticle size while maintaining consistency and high uniformity is of paramount importance for improving the efficacy of nanoparticle-based therapies and minimizing potential side effects. Although microfluidic technologies are widely used for reliable nanoparticle synthesis, they face challenges in meeting critical homogeneity requirements, mainly due to imperfect mixing efficiency. Furthermore, channel clogging during continuous operation presents a significant obstacle in terms of quality control, as it progressively impedes the mixing behavior necessary for consistent nanoparticle production for therapeutic delivery and complicates the scaling-up process. This study entailed the development of a 3D-printed novel micromixer embedded with hemispherical baffle microstructures, a dual vortex mixer (DVM), which integrates Dean vortices to generate two symmetrical counter-rotating intensified secondary flows. The DVM with a relatively large mixer volume showed rapid mixing characteristics even at a flow rate of several mL min^-1 and produced highly uniform lipids, liposomes, and polymer nanoparticles in a size range (50-130 nm) and polydispersity index (PDI) values below 0.15. For the evaluation of products, SARS-CoV-2 Spike mRNA-loaded lipid nanoparticles were examined to verify protein expression in vitro and in vivo using firefly luciferase (FLuc) mRNA. This showed that the performance of the system is comparable to that of a commercial toroidal mixer. Moreover, the vigorous in-situ dispersion of nanoparticles by harnessing the power of vortex physically minimizes the occurrence of aggregation, ensuring consistent production performance without internal clogging of a half-day operation and facilitating quality control of the nanoparticles at desired scales.

Messenger RNA (mRNA) vaccines represent a critical avenue for coronavirus disease 2019 (COVID-19) prevention. We developed a COVID-19 mRNA vaccine encoding a codon-optimized full-length ancestral spike (S) protein with a signal peptide, which employs our novel patented co-transcriptional 5′-capping reagent, SmartCap^®. From the screening capping library of SmartCap^®, an SC101 cap was selected to derive a novel mRNA vaccine, STP2104. An in vitro study of STP2104 incorporating SC101 revealed enhanced protein expression in both the cell lysate and culture medium, and an in vivo immunogenicity study revealed strong humoral and cell-mediated immune responses. STP2104 further displayed potent neutralizing activity in immunized mice as derived via the PRNT₅₀ assay using the wild-type virus. We evaluated the protection efficacy of STP2104 using human ACE2 transgenic mice immunized and challenged with SARS-CoV-2 to acquire the survival rate, virus titration, and histopathology study data. These studies proved that STP2104 is potent enough to induce protective immunity. A novel capping library screening (CLS) method was successfully utilized for exploring the optimal 5′-cap reagent, which improves S gene expression with mRNA stability. The clinical phase 1 studies of STP2104 will prove its safety, tolerability, and immunogenicity as well as the safety of the novel 5′-cap analogue SC101 in humans.

We have developed a 5′-capping library screening (CLS) method using over 30 different novel cap analogues. The optimal 5′-cap for the coronavirus disease 2019 (COVID-19) mRNA vaccine STP2104 was selected and applied. This is the first report to describe the proven safety of the novel cap analogue, SmartCap^® SC101, in humans and emphasize the importance of cap selection. STP2104 demonstrates safety, tolerability, and strong immune responses in humans. After confirming its safety through a GLP toxicity study, STP2104 was administered intramuscularly as a two-dose vaccine, separated by 28 days, in COVID-19-naive, healthy adult volunteers. In this multicenter, open-label, dose-escalation, phase I study with 30 participants (18 to 55 years of age), 15 individuals each were assigned to the low-dose (25 μg) and high-dose (50 μg) cohorts. The primary endpoints were the safety and immunogenicity in all cohorts. During the reporting period of the trial, no serious adverse events were reported. A plaque reduction neutralization test demonstrated an at least 21-fold increase in NAb titers from both cohorts when comparing pre-vaccination to 4-week post-second vaccination. These safety and NAb titer interim results support the efficiency and safety of SC101 and the STP2104 mRNA vaccine, including how STP2104 effectively induces NAb titers against SARS-CoV-2.

Background/Objectives: Messenger RNA (mRNA) vaccines have enabled rapid vaccine development and have become an important platform for pandemic preparedness; however, clinical evaluation of alternative co-transcriptional 5′ cap technologies remains limited. Here, we report the first-in-human safety, tolerability, and immunogenicity of STP2104, a SARS-CoV-2 mRNA vaccine incorporating the novel co-transcriptional 5′ cap analogue SmartCap^® SC101. Methods: In this Phase I, multicenter, open-label, dose-escalation study, 30 healthy adults with no known history of SARS-CoV-2 infection received two intramuscular doses of STP2104 at either 25 μg or 50 μg on Days 0 and 28. Safety and tolerability were evaluated, along with humoral and cellular immune responses, including neutralizing antibodies, binding antibodies, and antigen-specific IFN-γ ELISpot responses. All participants completed the planned two-dose vaccination schedule before early study termination. Results: STP2104 demonstrated an acceptable short-term safety and tolerability profile across both dose cohorts, with no treatment-emergent serious adverse events, Grade ≥ 3 treatment-emergent adverse events under the protocol-defined clinical severity criteria, or events leading to study discontinuation. However, one participant experienced a transient Grade 3 platelet-count laboratory abnormality, which was recorded in the Clinical Study Report (CSR) as a Grade 1 thrombocytopenia treatment-emergent adverse event. Neutralizing antibody titers increased following vaccination in both dose cohorts, reaching their post-boost maximum at Day 35 and remaining above baseline at Day 56. Binding-antibody concentrations also increased following vaccination, and antigen-specific IFN-γ ELISpot responses were detected in both dose cohorts. Conclusions: This first-in-human Phase I study shows that SmartCap^® SC101 can be incorporated into an mRNA–LNP vaccine construct for clinical evaluation. STP2104 had an acceptable short-term safety and tolerability profile and induced measurable humoral and cellular immune responses. These results support further clinical evaluation of SmartCap^® SC101-containing mRNA constructs with updated or alternative antigens.