The remarkable impact of mRNA vaccines on mitigating disease and improving public health has been amply demonstrated during the COVID-19 pandemic. Many new mRNA-based vaccine and therapeutic candidates are in development, yet the current reality of their stability limitations requires their frozen storage. Numerous challenges remain to improve formulated mRNA stability and enable refrigerator storage, and this review provides an update on developments to tackle this multi-faceted stability challenge. We describe the chemistry underlying mRNA degradation during storage and highlight how lipid nanoparticle (LNP) formulations are a double-edged sword: while LNPs protect mRNA against enzymatic degradation, interactions with and between LNP excipients introduce additional risks for mRNA degradation. We also discuss strategies to improve mRNA stability both as a drug substance (DS) and a drug product (DP) including the (1) design of the mRNA molecule (nucleotide selection, primary and secondary structures), (2) physical state of the mRNA-LNP complexes, (3) formulation composition and purity of the components, and (4) DS and DP manufacturing processes. Finally, we summarize analytical control strategies to monitor and assure the stability of mRNA-based candidates, and advocate for an integrated analytical and formulation development approach to further improve their storage, transport, and in-use stability profiles.

Coronavirus (SARS-CoV-2) as a global pandemic has attracted the attention of many scientific centers to find the right treatment. We expressed and purified the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein, and specific RBD aptamers were designed using SELEX method. RNAi targeting nucleocapsid phosphoprotein was synthesized and human lung cells were inoculated with aptamer-functionalized lipid nanoparticles (LNPs) containing RNAi. The results demonstrated that RBD aptamer having KD values of 0.290 nm possessed good affinity. Based on molecular docking and efficacy prediction analysis, siRNA molecule was showed the best action. LNPs were appropriately functionalized by aptamer and contained RNAi molecules. Antiviral assay using q-PCR and ELISA demonstrated that LNP functionalized with 35 µm Apt and containing 30 nm RNAi/ml of cell culture had the best antiviral activity compared to other concentrations. Applied aptamer in the nanocarrier has two important functions. First, it can deliver the drug (RNAi) to the surface of epithelial cells. Second, by binding to the SARS-CoV-2 spike protein, it inhibits the virus entrance into cells. Our data reveal an interaction between the aptamer and the virus, and RNAi targeted the virus RNA. CT scan and the clinical laboratory tests in a clinical case study, a 36-year old man who presented with severe SARS-CoV-2, demonstrated that inhalation of 10 mg Apt-LNPs-RNAi nebulized/day for six days resulted in an improvement in consolidation and ground-glass opacity in lungs on the sixth day of treatment. Our findings suggest the treatment of SARS-CoV-2 infection through inhalation of Aptamer-LNPs-RNAi.

: There is a global need for effective and affordable rabies vaccines, which is unmet by current vaccines due to limitations in their production capacities, required administration schedules, storage requirements, and cost. Many different experimental approaches previously used for bacterial and viral vaccines have been applied to rabies, but with variable success. One of the most promising new concepts is the use of messenger RNA (mRNA) in encoding the main rabies virus antigen, the envelope glycoprotein (RABV-G). CureVac has applied their proprietary technology platform for the production of mRNA to this problem, resulting in the rabies vaccine candidate CV7201. Following preclinical studies in mice and pigs showing that CV7201 could induce neutralizing immune responses that protected against rabies virus, different dosages and routes of administration of CV7201 were tested in a phase 1 human study. This clinical study proved that mRNA vaccination was safe and had an acceptable reactogenicity profile, but immune responses depended on the mode of administration, and they did not unequivocally support CV7201 for further development as a prophylactic vaccine with this particular formulation. Further, preclinical studies using RABV-G mRNA encapsulated in lipid nanoparticles (LNPs) showed an improved response in both mice and nonhuman primates, and these encouraging results are currently being followed up in clinical studies in humans. This review summarizes the recent advances in mRNA vaccines against rabies.

With Quality by Design (QbD), a systematic approach involving design and development of all production processes to achieve the final product with a predetermined quality, you work within a design space that determines the critical formulation and process parameters. Verification of the quality of the final product is no longer necessary. In the current study, the QbD approach was used in the preparation of lipid nanoparticle formulations to improve skin penetration of 5-Fluorouracil, a widely-used compound for treating non-melanoma skin cancer. 5-Fluorouracil-loaded lipid nanoparticles were prepared by the W/O/W double emulsion - solvent evaporation method. Artificial neural network software was used to evaluate the data obtained from the lipid nanoparticle formulations, to establish the design space, and to optimize the formulations. Two different artificial neural network models were developed. The limit values of the design space of the inputs and outputs obtained by both models were found to be within the knowledge space. The optimal formulations recommended by the models were prepared and the critical quality attributes belonging to those formulations were assigned. The experimental results remained within the design space limit values. Consequently, optimal formulations with the critical quality attributes determined to achieve the Quality Target Product Profile were successfully obtained within the design space by following the QbD steps.