Despite the usage of DNA vaccines, RNA-based vaccines have grown in popularity in the 21st century. Interestingly, mRNA vaccine technology purely depends on the genetic code of the virus; since the SARS-CoV-2 genetic sequence was published ^[1][58], virally generated self-amplifying RNA and non-replicating mRNA are being used abundantly for vaccine development. Indeed, during the COVID-19 pandemic, mRNA vaccines have become the only vaccine source available to combat the rapid spread of disease. While multiple pharmaceutical companies have investigated several vaccine technologies against COVID-19, the FDA has approved two vaccines (first for emergency use and then for full approval), Pfizer/BioNTech’s and Moderna’s Spikevax (Table 12) ^[2][59]. Both use the complete sequence of spike and receptor-binding domains of SARS-CoV-2 as target antigens. However, mRNA-based preclinical studies have been reported since 1990. Its poor stability as naked mRNA and tremendous capacity to induce an innate immune response (particularly double-stranded RNA) has halted the progress of RNA-based therapeutics. The emergence of advanced nanotechnologies for the protection and targeted delivery of water-soluble molecules have shined a light on developing the LNP-mRNA vaccines. Nucleic acid delivery to the cytosol, without hampering the cell wall integrity, and degradation by nucleases is a rate limiting step for targeted delivery. Despite mechanical and viral particle-mediated targeted delivery, chemical based targeted delivery via LNPs have been the ease of the process and have been less toxic, with less immunogenicity. During the start of the decade, several clinical trials have been initiated on mRNA therapeutics, which cover a range of different types of diseases, such as cancer, infectious diseases, protein replacement therapy, and others. Correspondingly, promising results have been shown in terms of disease alleviation, reduction of pathogenicity, and others. Interestingly, several diseases or disorders are targeted by using mRNA therapeutics. For the proof-of-concept studies, in vitro transcription (IVT)-produced mRNA is more suitable (Figure 15). To characterize the efficiency of mRNA in vitro, several transfection reagents are available to facilitate mRNA translation and targeted protein production, although they are not as efficient as targeted delivery systems. The Figure 15 shows the most successful mRNA vaccines. As explained earlier, a suitable (perfect mix) vehicle is needed for the mRNA vaccine delivery. LNPs have become the one of the most characterized mRNA delivery vehicles.

Using lipid-encapsulated forms of sequence-optimized mRNA vaccines has shown strong immunity (both in preclinical and clinical) against infectious disease targets like the flu virus, Zika virus, rabies virus, and others, especially in the last few years ^[3][60]. Of note, the LNPs act as potential vaccine adjuvants ^[2][59], which helps in shaping antigen-specific immunity ^[4][61]. Despite several components of the LNP, ionizable lipids have a prominent role, including forming LNPs and the targeted delivery of mRNA into the cytosol. The ionizable lipid imparts the neutral charge to the LNPs at physiological pH and ionizes at lower pH in the endosome/lysosome, followed by the delivery of intact mRNA to the cytoplasm, where it can then be translated into the encoded protein. Despite mRNA, LNPs also induce the innate immune signaling pathways that aid in the adaptive immune responses. Similar to other potential adjuvants, LNPs (based on the composition) also induce the activation of several signaling pathways, including nuclear factor kappa B, interferons, inflammasome activation, and other ^[5][46].

In the future, it will be essential to compare and understand the immune pathways activated by different mRNA vaccine platforms, improve current approaches based on these mechanisms, and start new clinical trials against more disease targets.

⁺ T cells) and release several cytokines and chemokines, which further help in the shaping the antigen-specific immune responses, including B cell differentiation and plasma cell development ^[13][69]. The two successful vaccines (Pfizer/BioNTech and Moderna’s Spikevax) have confirmed this mechanism in both preclinical and clinical studies (Figure 26) ^[14][70].

Even though the needle-free route of administration is long awaited ^[6][7][8][62,63,64], mRNA encapsulated by LNPs is administered mostly by the intramuscular route, and several study results support this route of administration ^[9][10][11][65,66,67]. Upon injection, local innate immune cells are recruited to the site of injection, followed by the uptake of LNPs by innate immune cells ^[12][68]. Once the LNPs reach the cytosol, lower pH enables the LNPs to release mRNA, followed by the translation of targeted antigens (Figure 15). The antigens are presented to the T helper cells (CD4