Sodium alginate coating simultaneously increases the biosafety and immunotherapeutic activity of the cationic mRNA nanovaccine.

The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.

A lipid-based LMP2-mRNA vaccine to treat nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a serious and highly invasive epithelial malignancy that is closely associated with Epstein-Barr virus (EBV). Due to the lack of therapeutic vaccines for NPC, we selected EBV latent membrane protein 2 (LMP2) as a preferable targeting antigen to develop a lipid-based LMP2-mRNA (mLMP2) vaccine. Full-length mLMP2 expressing LMP2 was first synthesized using an in vitro transcription method and then encapsulated into (2,3-dioleacyl propyl) trimethylammonium chloride (DOTAP)-based cationic liposomes to obtain the mRNA vaccine (LPX-mLMP2). The cell assays showed that the antigen-presenting cells were capable of highly efficient uptake of LPX-mLMP2 and expression of LMP2. LMP2 could subsequently be presented to form the peptide-major histocompatibility complex (pMHC). Furthermore, LPX-mLMP2 could accumulate in the spleen, express antigens, promote the maturation of dendritic cells and stimulate antigen-specific T-cell responses in vivo. It dramatically inhibited the tumor growth of the LMP2-expressing tumor model after three doses of vaccination. Additionally, the proliferation of antigen-specific T cells in the tumor site made a good sign for the promise of mRNA vaccines in virus-induced cancer. Overall, we provided a newly developed antigen-encoding mRNA vaccine with advantages against NPC. We also demonstrated that mRNA vaccines are attractive candidates for cancer immunotherapy. Electronic Supplementary Material: Supplementary material (methods of cytotoxicity assay, LMP2 expression, hemolysis test, the results of purity and maturity of BMDCs, LMP2 expression, and evaluation of T cells in lymph nodes and gating strategy for CTLs) is available in the online version of this article at 10.1007/s12274-022-5254-x.

A novel heterologous receptor-binding domain dodecamer universal mRNA vaccine against SARS-CoV-2 variants.

There are currently approximately 4,000 mutations in the SARS-CoV-2 S protein gene and emerging SARS-CoV-2 variants continue to spread rapidly worldwide. Universal vaccines with high efficacy and safety urgently need to be developed to prevent SARS-CoV-2 variants pandemic. Here, we described a novel self-assembling universal mRNA vaccine containing a heterologous receptor-binding domain (HRBD)-based dodecamer (HRBDdodecamer) against SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), Gamma (B.1.1.28.1), Delta (B.1.617.2) and Omicron (B.1.1.529). HRBD containing four heterologous RBD (Delta, Beta, Gamma, and Wild-type) can form a stable dodecameric conformation under T4 trimerization tag (Flodon, FD). The HRBDdodecamer -encoding mRNA was then encapsulated into the newly-constructed LNPs consisting of a novel ionizable lipid (4N4T). The obtained universal mRNA vaccine (4N4T-HRBDdodecamer) presented higher efficiency in mRNA transfection and expression than the approved ALC-0315 LNPs, initiating potent immune protection against the immune escape of SARS-CoV-2 caused by evolutionary mutation. These findings demonstrated the first evidence that structure-based antigen design and mRNA delivery carrier optimization may facilitate the development of effective universal mRNA vaccines to tackle SARS-CoV-2 variants pandemic.

Lipid Nanoparticle Delivery System for mRNA Encoding B7H3-redirected Bispecific Antibody Displays Potent Antitumor Effects on Malignant Tumors.

The therapeutic use of bispecific T-cell engaging (BiTE) antibodies has shown great potential for treating malignancies. BiTE can simultaneously engage CD3ε on T cells and tumor antigen on cancer cells, thus exerting an effective antitumor effect. Nevertheless, challenges in production, manufacturing, and short serum half-life of BiTE have dampened some of the promise and impeded the pace of BiTE-based therapeutics to combat diseases. Nowadays, in vitro-transcribed mRNA has achieved programmed production, which is more flexible and cost-effective than the traditional method of producing recombinant antibody. Here, the authors have developed a BiTE-based mRNA treatment by encapsulating mRNA encoding B7H3×CD3 BiTE into a novel ionizable lipid nanoparticles (LNPs). The authors have found that LNPs have high transfection efficiency, and the hepatosplenic targeting capability of produce high concentrations of BiTE. Above all, a single intravenous injection of BiTE mRNA-LNPs could achieve high levels of protein expression in vivo and significantly prolonged the half-life of the BiTE, which can elicit robust and durable antitumor efficacy against hematologic malignancies and melanoma. Therefore, their results suggested that the therapeutic strategy based on mRNA expression of B7H3×CD3 BiTE is of potential research value and has promising clinical application prospects.

Manganese-coordinated mRNA vaccines with enhanced mRNA expression and immunogenicity induce robust immune responses against SARS-CoV-2 variants.

It is urgent to develop more effective mRNA vaccines against the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants owing to the immune escape. Here, we constructed a novel mRNA delivery system [IC8/Mn lipid nanoparticles (IC8/Mn LNPs)]with high immunogenicity, via introducing a stimulator of interferon genes (STING) agonist [manganese (Mn)] based on a newly synthesized ionizable lipid (IC8). It was found that Mn can not only promote maturation of antigen-presenting cells via activating STING pathway but also improve mRNA expression by facilitating lysosomal escape for the first time. Subsequently, IC8/Mn LNPs loaded with mRNA encoding the Spike protein of SARS-CoV-2 Delta or Omicron variant (IC8/Mn@D or IC8/Mn@O) were prepared. Both mRNA vaccines induced substantial specific immunoglobulin G responses against Delta or Omicron. IC8/Mn@D displayed strong pseudovirus neutralization ability, T helper 1-biased immune responses, and good safety. It can be concluded that IC8/Mn LNPs have great potential for developing Mn-coordinated mRNA vaccines with robust immunogenicity and good safety.

mRNA Vaccines Against SARS-CoV-2 Variants Delivered by Lipid Nanoparticles Based on Novel Ionizable Lipids.

SARS-CoV-2 variants are now still challenging all the approved vaccines, including mRNA vaccines. There is an urgent need to develop new generation mRNA vaccines with more powerful efficacy and better safety against SARS-CoV-2 variants. In this study, a new set of ionizable lipids named 4N4T are constructed and applied to form novel lipid nanoparticles called 4N4T-LNPs. Leading 4N4T-LNPs exhibit much higher mRNA translation efficiency than the approved SM-102-LNPs. To test the effectiveness of the novel delivery system, the DS mRNA encoding the full-length S protein of the SARS-CoV-2 variant is synthesized and loaded in 4N4T-LNPs. The obtained 4N4T-DS mRNA vaccines successfully trigger robust and durable humoral immune responses against SARS-CoV-2 and its variants including Delta and Omicron. Importantly, the novel vaccines have higher RBD-specific IgG titers and neutralizing antibody titers than SM-102-based DS mRNA vaccine. Besides, for the first time, the types of mRNA vaccine-induced neutralizing antibodies are found to be influenced by the chemical structure of ionizable lipids. 4N4T-DS mRNA vaccines also induce strong Th1-skewed T cell responses and have good safety. This work provides a novel vehicle for mRNA delivery that is more effective than the approved LNPs and shows its application in vaccines against SARS-CoV-2 variants.

mRNA-based therapeutics: powerful and versatile tools to combat diseases.

The therapeutic use of messenger RNA (mRNA) has fueled great hope to combat a wide range of incurable diseases. Recent rapid advances in biotechnology and molecular medicine have enabled the production of almost any functional protein/peptide in the human body by introducing mRNA as a vaccine or therapeutic agent. This represents a rising precision medicine field with great promise for preventing and treating many intractable or genetic diseases. In addition, in vitro transcribed mRNA has achieved programmed production, which is more effective, faster in design and production, as well as more flexible and cost-effective than conventional approaches that may offer. Based on these extraordinary advantages, mRNA vaccines have the characteristics of the swiftest response to large-scale outbreaks of infectious diseases, such as the currently devastating pandemic COVID-19. It has always been the scientists' desire to improve the stability, immunogenicity, translation efficiency, and delivery system to achieve efficient and safe delivery of mRNA. Excitingly, these scientific dreams have gradually been realized with the rapid, amazing achievements of molecular biology, RNA technology, vaccinology, and nanotechnology. In this review, we comprehensively describe mRNA-based therapeutics, including their principles, manufacture, application, effects, and shortcomings. We also highlight the importance of mRNA optimization and delivery systems in successful mRNA therapeutics and discuss the key challenges and opportunities in developing these tools into powerful and versatile tools to combat many genetic, infectious, cancer, and other refractory diseases.