The promise of mRNA vaccines: a biotech and industrial perspective

“3 SAM differs with the inclusion of genetic replication machinery derived from positive-stranded mRNA viruses, most commonly from alphaviruses such as Sindbis and Semliki-Forest viruses.”

“First, the purity of the mRNA is a crucial determinant of yields, and it is known that the DNA-dependent RNA polymerases yield smaller oligoribonucleotide impurities as a result of abortive initiation events, 11 as well as double-stranded (ds) RNA generated by self-complementary 3′ extension, 12 which can result in ty…”

“32, 33 This has been an intense area of research in the field, for which lipid nanoparticle (LNP) formulations are currently emerging as a leading category.”

“49, 50 Additional sites within the mRNA construct are available for optimization as well.”

“[41] [42] [43] [44] Much of the focus of the continued development of such LNP carrier systems involves optimization of the ionizable lipid component, with particular focus on the acid dissociation constant (pKa) and fusogenic properties (both of the ionizable component as well as helper lipid[s]), which have been dem…”

“[45] [46] [47] [48] Next-generation LNPs may include specific targeting motifs for homing and uptake by professional antigen-presenting cells, such as dendritic cells (DC).”

“20 Manufacturers of mRNA vaccines pay careful attention to the choice of enzyme and reaction conditions, in order to catalyze the highest percentage of cap formation.”

“[45] [46] [47] [48] Next-generation LNPs may include specific targeting motifs for homing and uptake by professional antigen-presenting cells, such as dendritic cells (DC).”

“[24] [25] [26] It has been demonstrated that successful protein production, minimal undesired inflammatory responses, and systemic adaptive immune responses could be achieved preclinically by using unmodified mRNA 27 through a combination of optimizing the coding sequence and removal of any unwanted inflammatory impur…”

“[24] [25] [26] It has been demonstrated that successful protein production, minimal undesired inflammatory responses, and systemic adaptive immune responses could be achieved preclinically by using unmodified mRNA 27 through a combination of optimizing the coding sequence and removal of any unwanted inflammatory impur…”

“62, 63 Whether this lack of translation is a function of mRNA not having optimized quality attributes, suboptimal delivery or an inherent limitation of preclinical or in vitro translational models is not known.”

“32, 33 This has been an intense area of research in the field, for which lipid nanoparticle (LNP) formulations are currently emerging as a leading category.”

“[45] [46] [47] [48] Next-generation LNPs may include specific targeting motifs for homing and uptake by professional antigen-presenting cells, such as dendritic cells (DC).”

“63, 69 This indicates that much formulation work is still needed to achieve sufficient immunological potency of different vaccine candidates, while maintaining acceptable tolerability-but we can be encouraged by incremental progress to date.”

“[24] [25] [26] It has been demonstrated that successful protein production, minimal undesired inflammatory responses, and systemic adaptive immune responses could be achieved preclinically by using unmodified mRNA 27 through a combination of optimizing the coding sequence and removal of any unwanted inflammatory impur…”

“64 The current focus from a clinical perspective is to optimize the benefit (immunogenicity and efficacy) while reducing the risk (safety) profile of a candidate mRNA vaccine by optimizing the quality attributes that dictate expression and/or augmenting delivery.”

“For example, optimization of guanine and cytosine (GC) content can have a significant impact, 23 and has been well established with DNA vaccines.”

“[41] [42] [43] [44] Much of the focus of the continued development of such LNP carrier systems involves optimization of the ionizable lipid component, with particular focus on the acid dissociation constant (pKa) and fusogenic properties (both of the ionizable component as well as helper lipid[s]), which have been dem…”

“63, 69 This indicates that much formulation work is still needed to achieve sufficient immunological potency of different vaccine candidates, while maintaining acceptable tolerability-but we can be encouraged by incremental progress to date.”

“21, 22 Last, codon optimization and modification of nucleotides have contributed to translation efficiency.”

“49, 50 Additional sites within the mRNA construct are available for optimization as well.”

“[16] [17] [18] In addition, 2-O′-unmethylated capped RNA can be sequestered by cellular IFN-induced proteins with tetratricopeptide repeats (IFIT1) that prevent the initiation of translation, 19 or detected by the cytoplasmic RNA sensor MDA5.”

“9 Enzymatic addition of the cap can be accomplished by using guanylyl transferase and 2′-O-methyltransferase to yield a Cap 0 ( N7Me GpppN) or Cap 1 ( N7Me GpppN 2′-OMe ) structure, respectively, while the poly-A tail can be achieved through enzymatic addition via poly-A polymerase.”

“21, 22 Last, codon optimization and modification of nucleotides have contributed to translation efficiency.”

“[55] [56] [57] The combinations of all of these areas for optimization are near limitless, and success within any of these parameters can allow for beneficial effects and can provide novel approaches for vaccines to successfully prevent disease.”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“[41] [42] [43] [44] Much of the focus of the continued development of such LNP carrier systems involves optimization of the ionizable lipid component, with particular focus on the acid dissociation constant (pKa) and fusogenic properties (both of the ionizable component as well as helper lipid[s]), which have been dem…”

“[55] [56] [57] The combinations of all of these areas for optimization are near limitless, and success within any of these parameters can allow for beneficial effects and can provide novel approaches for vaccines to successfully prevent disease.”

“1 mRNA constructs can also be used to express potent monoclonal antibodies for novel immunoprophylaxis.”

“[16] [17] [18] In addition, 2-O′-unmethylated capped RNA can be sequestered by cellular IFN-induced proteins with tetratricopeptide repeats (IFIT1) that prevent the initiation of translation, 19 or detected by the cytoplasmic RNA sensor MDA5.”

“[41] [42] [43] [44] Much of the focus of the continued development of such LNP carrier systems involves optimization of the ionizable lipid component, with particular focus on the acid dissociation constant (pKa) and fusogenic properties (both of the ionizable component as well as helper lipid[s]), which have been dem…”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“First, the purity of the mRNA is a crucial determinant of yields, and it is known that the DNA-dependent RNA polymerases yield smaller oligoribonucleotide impurities as a result of abortive initiation events, 11 as well as double-stranded (ds) RNA generated by self-complementary 3′ extension, 12 which can result in ty…”

“2 Here we review the state of the art in mRNA constructs and delivery technologies for the prevention of infectious diseases, and a review of pertinent topics to the biotechnology and pharmaceutical industries.”

“28, 29 Ultimately, comparative immunogenicity between these approaches require studies that control all potential factors, including the delivery system.”

“[55] [56] [57] The combinations of all of these areas for optimization are near limitless, and success within any of these parameters can allow for beneficial effects and can provide novel approaches for vaccines to successfully prevent disease.”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“[52] [53] [54] Optimization of the carrier system can provide significant benefit as well.”

“The manufacturing process begins with the generation of a plasmid DNA (pDNA) containing a DNA-dependent RNA polymerase promoter, such as T7, 6 and the corresponding sequence for the mRNA construct.”

“[45] [46] [47] [48] Next-generation LNPs may include specific targeting motifs for homing and uptake by professional antigen-presenting cells, such as dendritic cells (DC).”

“4, 5 Generally, the ORF encoding viral structural proteins is replaced by the selected transcript of interest, and the viral RNA-dependent RNA polymerase is retained to direct cytoplasmic amplification of the replicon construct.”

“30, 31 In addition to optimizing an mRNA construct, and of quintessential importance, is the delivery of the mRNA vaccine from the bolus at the injection site into the cytoplasm of cells for the initiation of translation.”

“58 Therefore, safety and tolerability may limit multi-antigen approaches, and here translational sciences are crucial for development.”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“The addition of the 5′ cap and the 3′ poly(A) tail can be achieved during the in vitro transcription step 7, 8 or enzymatically after transcription.”

“[52] [53] [54] Optimization of the carrier system can provide significant benefit as well.”

“59 Systems biology techniques can help in the understanding of fine differences between various NRM and SAM vaccine sequences, as well as serving as useful tools to frame sequence optimizations during iterative development schemes.”

“51 Improved stability of mRNA can also be achieved through various modifications of the triphosphate bridge within the cap structure.”

“[16] [17] [18] In addition, 2-O′-unmethylated capped RNA can be sequestered by cellular IFN-induced proteins with tetratricopeptide repeats (IFIT1) that prevent the initiation of translation, 19 or detected by the cytoplasmic RNA sensor MDA5.”

“Thus, early-phase clinical trials need to be designed in a way to appropriately capture the inflammatory component intrinsic to all mRNA vaccines, given that several intracellular innate immune response sensors are activated by RNA. 65 Elements include measuring administration site reactions such as pain, tenderness-a…”

“4, 5 Generally, the ORF encoding viral structural proteins is replaced by the selected transcript of interest, and the viral RNA-dependent RNA polymerase is retained to direct cytoplasmic amplification of the replicon construct.”

“[16] [17] [18] In addition, 2-O′-unmethylated capped RNA can be sequestered by cellular IFN-induced proteins with tetratricopeptide repeats (IFIT1) that prevent the initiation of translation, 19 or detected by the cytoplasmic RNA sensor MDA5.”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“[52] [53] [54] Optimization of the carrier system can provide significant benefit as well.”

“60 Further, the use of functional assessments, like human challenge models, 61 where the immunologic profiles of the participants and the specific details of the challenge strain(s) are well characterized-an approach mostly used to date in the evaluation of more traditional vaccinesprovides an important and powerful m…”

“[34] [35] [36] There have been numerous examples of successful delivery of mRNA by using LNPs for therapeutic [37] [38] [39] [40] as well as vaccine applications.”

“After identification of the protective protein antigen(s) and sequencing the corresponding gene(s), the mRNA can be made within weeks (Jackson et al., 2020).”