Lipid particles for delivering RNA vaccines

Abstract

Disclosed herein are RNA lipid particles comprising a compound of Formula (I) and their use as vaccines or medicaments.

Description

LIPID PARTICLES FOR DELIVERING RNA VACCINESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application Nos. 63 / 722,744, filed November 20, 2024, U.S. 63 / 763,810, filed February 26, 2025, and U.S. 63 / 814,988, filed May 30, 2025. The entire contents of the above-identified applications are hereby fully incorporated herein by reference.SEQUENCE LISTING

[0002] This application contains a sequence listing filed in electronic form as an xml file entitled BROD-6060WP_ST26, created on November 13, 2025, and having a size of 2,256 bytes. The content of the sequence listing is incorporated herein in its entirety.TECHNICAL FIELD

[0003] The subject matter disclosed herein is generally directed to novel lipid particles for delivering nucleic acids such as mRNA. Also provided are methods for making and using lipid particles to deliver nucleic acids such as mRNA.BACKGROUND

[0004] Infectious diseases continue to pose significant global health challenges despite advances in vaccine development. Emerging and re-emerging pathogens, including those designated as World Health Organization R&D Blueprint priority pathogens, present ongoing threats to public health security. Many high-consequence pathogens lack licensed vaccines or effective therapeutic interventions, creating critical gaps in pandemic preparedness and global health protection.

[0005] Messenger RNA (mRNA)-based vaccines represent a transformative approach to rapid vaccine development, offering significant advantages over traditional vaccine platforms. Unlike conventional subunit vaccines that rely on recombinantly produced antigenic proteins requiring complex bacterial fermentation, cell culture, and purification processes, mRNA vaccines enable direct in vivo antigen production. This approach allows for proper post- translational modification and natural antigen presentation while bypassing costly, timeintensive manufacturing steps. The modular nature of mRNA vaccine technology facilitates rapid adaptation to different antigens, enabling a swift response to emerging threats.Additionally, mRNA vaccines provide only transient antigen expression without genomic integration, and effectively stimulate both cellular and humoral immune responses.

[0006] However, current mRNA vaccine platforms face significant technical limitations that constrain their broader therapeutic application. The translation efficiency of synthetic mRNAs is often suboptimal, limiting antigen expression and often necessitating multiple booster doses to achieve protective immunity. Reactogenicity remains a persistent challenge, stemming from both mRNA backbone components and lipid nanoparticle (LNP) formulations, potentially causing excessive inflammatory responses or hypersensitivity reactions. Furthermore, off-target translation events and uncontrolled immune activation can compromise both safety and efficacy. The inherent instability of RNA molecules and the absence of natural cellular uptake mechanisms necessitate the use of sophisticated delivery systems.

[0007] Optimizing mRNA vaccine performance requires integrated engineering of multiple molecular and nanomaterial parameters. Similarly, LNP composition, particularly the selection of ionizable lipids, profoundly affects mRNA encapsulation, cellular delivery, and immune activation patterns. Achieving optimal balance between immunogenicity and tolerability demands systematic optimization of these interconnected components.

[0008] Crimean-Congo hemorrhagic fever virus (CCHFV) exemplifies the urgent need for advanced vaccine platforms against high-consequence pathogens. This tick-borne orthonairovirus causes severe hemorrhagic disease with case fatality rates reaching 40% across endemic regions in Africa, Asia, Eastern Europe, and the Middle East. Climate change and the expansion of arthropod vector ranges have facilitated geographic spread, driving increased incidence in previously unaffected regions. Despite its broad distribution and epidemic potential, no licensed vaccines or specific antiviral therapies are available, and CCHFV remains a WHO priority pathogen requiring immediate development of countermeasures.

[0009] The development of improved mRNA vaccine delivery systems remains constrained by fundamental challenges in achieving efficient in vivo RNA delivery while maintaining appropriate immune balance. There is a compelling need for advanced lipid particle formulations that address these core limitations through systematic molecular and nanomaterial engineering, enabling broader therapeutic applications across diverse pathogen targets and clinical indications.SUMMARY

[0010] In certain example embodiments disclosed herein, lipid particles comprising a compound of Formula Ior a pharmaceutically acceptable salt thereof, whereinA1and A2are each independently unsubstituted Ci-C] ? alkylene.

[0011] Also disclosed herein is a lipid particle comprising the compound heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2-hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof.

[0012] In an example embodiment, disclosed herein is a pharmaceutical composition comprising the compound heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2- hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof. In one embodiment, one or more excipients are selected from neutral lipids, steroids, and polymer-conjugated lipids.

[0013] In certain example embodiments, disclosed herein is a method for raising an immune response comprising administering the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0014] Also disclosed herein is a method of prevention or treatment of cancer or tumor diseases, infectious diseases, allergies, or autoimmune diseases or disorders related thereto, comprising administering the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0015] Also disclosed herein is a method of administering a therapeutic agent to a subject, comprising providing the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0016] Also disclosed herein is a method for expressing a protein in a subject, comprising administering the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0017] Also disclosed herein is a method for vaccinating a subject against a viral pathogen, comprising administering the lipid particle or pharmaceutical composition of the invention to a subject.

[0018] Also disclosed herein is a method for vaccinating a subject against a bacterial pathogen comprising administering the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0019] Also disclosed herein is a method of preventing or treating cancer, comprising administering the lipid particle of the invention or the pharmaceutical composition of the invention to a subject.

[0020] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those with ordinary skill in the art upon considering the following detailed description of example embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0022] FIGS. 1A and IB - show a mammalian expression system in HEK293.

[0023] FIGS. 2A and 2B - show a bacterial expression system in rosetta bacteria.

[0024] FIGS. 3A-3C - show purification of a representative protein using an AKTA Pure chromatography system.

[0025] FIG. 4 - shows an amp-resistant polyA-plasmid template for a nucleocapsid.

[0026] FIG. 5 - shows nucleocapsid mRNA IVT synthesis.

[0027] FIG. 6 - shows different UTRs with nucleocapsid mRNA.

[0028] FIG. 7 - shows in vitro expression of the nucleocapsid in HEK293T cells.

[0029] FIGS. 8A-8C - characterizes the CCHF-LP’s pKa, T-LP’s pKa, and ACTA-LP’s pKa.

[0030] FIG. 9 - shows an exemplary immunization schedule.

[0031] FIGS. 10A-10C - are bar graphs showing cytokine release from CD8+ cells after 8-hour stimulation with five pg / ml of a nucleocapsid (200 pL / well).

[0032] FIGS. 11A-11E - are bar graphs showing cytokine release from CD4+ cells after 8-hour stimulation with five pg / ml of a nucleocapsid (200 pL / well).

[0033] FIGS. 12A and 12B - are bar graphs illustrating the results of an enzyme-linked immunosorbent spot (ELISpot) assay using bacterial-derived IL-2 or IFN-y proteins, following 24-hour stimulation with 5 pg / ml of IL-2 or IFN-y protein (200 pl / well).

[0034] FIGS 13A and 13B - are bar graphs illustrating the results of an ELISpot assay using HEK293 cell-derived IL-2 or IFN-y proteins, following 24-hour stimulation with 5 pg / ml of IL-2 or IFN-y protein (200 pl / well).

[0035] FIG 14 - shows an exemplary synthetic mRNA construct used for gene expression.

[0036] FIG. 15 - shows an agarose gel electrophoresis image assessing the integrity and presence of capped mRNA constructs, including (1) nucleocapsid mRNA, (2) mutant nucleocapsid mRNA, (3) mucin-gp38 mRNA, and (4) mucin-mutant-gp38 mRNA. The RiboRuler High Range RNA Ladder (ThermoFisher) serves as a molecular size reference on the left.

[0037] FIG. 16A-16B - is a bar graph showing the Z-Average (hydrodynamic diameter in nanometers, nm) of different mRNA formulations, including nucleocapsid, mutant nucleocapsid, mucin-gp38, and mucin-deglycosylated gp38.

[0038] FIG. 17A-17D - is a scatter plot showing the Poly dispersity Index (PDI) for different mRNA formulations, including nucleocapsid, mutant nucleocapsid, mucin-gp38, and mucin-deglycosylated gp38.

[0039] FIG. 18 - is a crystal structure showing nucleocapsid modification. Highly Conserved 266DEVD269 Motif of N: Cleavage Site for Caspase 3.

[0040] FIG. 19A - shows mucin-gp38 modification by removal of N-glycosylation sites in gp38 sequence.

[0041] FIG 19B - shows the amino acid sequence of mucin-gp38 and its N-glycosylation mutant used in the study; this sequence is provided as SEQ ID NO: 1 in the accompanying sequence listing.

[0042] FIG 19C - shows the predicted N-glycosylation sites.

[0043] FIG. 20A-20C - shows the nucleocapsid Western blot protein expression inHEK293, C2C12, and HepG2 cells following an 18-hour transfection. Protein was collected from cell lysates.

[0044] FIG. 21A-21C - shows the mucin-gp38 Western blot protein expression in HEK293, C2C12, and HepG2 cells following an 18-hour transfection. Protein was collected from supernatant.

[0045] FIGS. 22A-22D - show particle size distribution (in nanometers, nm) of distinct mRNA formulations, as determined by Dynamic Light Scattering (DLS) under steady-state conditions. Scattering intensity (%) is plotted against hydrodynamic diameter (d.nm) on a logarithmic scale is plotted against, reflecting the relative contribution of particle populations within each sample.

[0046] FIG. 23 - shows an exemplary in vivo experiment using 8-week-old female C57BL / 6J mice. The study includes five experimental groups: Nucleocapsid (n=8), Mutant- Nucleocapsid (n=8), Mucin-gp38 (n=8), Mucin-Deglyco-gp38 (n=8), and PBS control (n=5).

[0047] FIG. 24 - is a scatter plot showing the results of an ELISPOT assay, quantifying IFN-gamma-producing cells (measured as spot-forming units per 1.5x 106cells) across different experimental groups.

[0048] FIG. 25 - is a scatter plot showing the results of an ELISPOT assay, measuring IL- 4-producing cells, quantified as IL-4 spots per l.5>< 106cells across different experimental groups.

[0049] FIG. 26 - shows the immune response pathway following viral antigen uptake and the subsequent differentiation of immune cells leading to antibody production.

[0050] FIG. 27 - shows an exemplary flow cytometry gating strategy used for the identification and characterization of lymphocyte populations, including B cells, germinalcenter (GC) B cells, antigen-positive cells, CD4+ T cells, and T follicular helper (Tfh) cells. Each panel represents a sequential gating step to isolate specific immune cell subsets based on size, viability, and marker expression.

[0051] FIG. 28 - is a scatter plot showing the percentage of T follicular helper (Tfh) cells in lymph nodes across different experimental groups.

[0052] FIG. 29 - is a scatter plot showing the percentage of germinal center (GC) B cells in lymph nodes across different experimental groups.

[0053] FIG. 30 - is a scatter plot showing the percentage of antigen-positive germinal center (GC) B cells in lymph nodes across different experimental groups.

[0054] FIG. 31 - shows the IgM antibody response against the nucleocapsid antigen, measured using an in-house ELISA assay with a 1 :50 dilution of serum samples. Optical density (OD) at 450 nm (OD450) represents the level of IgM antibodies detected in each sample.

[0055] FIG. 32 - shows the IgG antibody response against the nucleocapsid antigen, measured using an in-house ELISA assay with a 1 :50 dilution of serum samples. Optical density (OD) at 450 nm (OD450) represents the level of IgM antibodies detected in each sample.

[0056] FIG. 33A-33C - compares the in vitro immunogenicity of BP- 104 to SM-102 and ALC-0315 when formulated to carry Nmut mRNA. A). Nmut mRNAs were encapsulated by LNPs containing BP- 104, SM-102 and ALC-0315. These mRNA / LNPs in different doses were given to IFN-a / p Reporter HEK 293 Cells and the IFN-I fold change to negative control (PBS) were documented. Empty LNPs were also included (iLNP-BP104, iLNP-SM102 and iLNP- ALC-0315) in the assay. As shown, all mRNA / LNP constructs have the potential to stimulate the IFN-I responses after 24 hours measured by SEAP. In addition, iLNPs triggered this response showing their adjuvant potential. B). These constructs also were tested in THPl-Dual cells to see their potential in stimulating the innate immunity by activating the NF-KB pathway and as demonstrated here, all mRNA / LNPs and iLNPs were able to activate this pathway. No significant differences were found between the groups. C). In this experiment, isolated and antigen-pulsed (Nmut-BP104, Nmut-SM102, and Nmut-ALC0315) CDl lc+dendritic cells were co-cultured with naive CD4+T cells from the spleen of C57BL / 6 mice for 4 days, and the intracellular cytokines were measured after 5-hour treatment of the cells with Brefeldin A. As shown, all three constructs activated CD4+ cells, as evidenced by elevated levels of IFN-gamma, TNF-alpha, and IL-17A. Nmut-ALC0315 had a significantly higher level of these cytokines than the other two groups. Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparisons test. Significant differences are shown as p-values.

[0057] FIG. 34A-34D - shows the immunogenicity of ACRA capped nucleocapsid mRNAs with different UTR regions in the C57BL / 6 mice model in a booster regimen. A) In this experiment, 8-week-old female C57BL / 6 mice were immunized with 10 pg of mRNA / LNPs at days 0 and 14, and blood and spleens were collected for ELISA and intracellular cytokine staining. B) The splenocytes were isolated from immunized mice on day 28 and stimulated for 5 hours with 10 pg / ml of nucleocapsid, then stained for intracellular cytokines IFN-gamma, IL-2, and TNF-alpha in CD8+ cells. As demonstrated, all these constructs significantly stimulated the production of all these cytokines compared to the negative control. No significant differences were observed among these three vaccine candidates, except for TNF-alpha, in which the ACTA1 construct showed a significantly higher level than the other two mRNA / LNP groups. C) Intracellular cytokines were measured in CD4+ cells, including IFN-gamma, IL-2, IL-4, TNF-alpha, and IL-17A. As with CD8+ cells, all constructs produced these cytokines, and no significant differences were observed among the three constructs, except for IL-17A, for which the ACTA-1 construct showed a significant difference. D) The splenocytes from immunized mice were collected and stimulated for 24 hours with 10 pg / ml of nucleocapsid to perform ELISPOT. As documented, all three vaccine candidates significantly increased the secretion of IFN-gamma and IL-2 cytokines compared to negative control mice. A significant difference was observed among the three vaccine candidates, with the CCHFV and ACTA-1 UTRs showing a higher response than the T-UTR group. E) shows in-house ELISA analysis of total IgG antibody responses against nucleocapsid protein. Positive antibody responses were detected in three of four mice in both vaccine groups at a 1 :50 dilution. Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparisons test. Significant differences are shown as p-values.

[0058] FIG. 35A-35D - shows immunogenicity of AG capped nucleocapsid, Nmut, GP38 and GP38Aglyc mRNAs with CCHFV UTR region in C57BL / 6 mice model in a booster regime. A) The same immunization schedule was used using AG capped mRNA / LNPs (nucleocapsid, Nmut, GP38, and GP38Aglyc). Blood, lymph node, and spleen were collected on day 28 for downstream immunological analyses, including ELISA, GC B and Tfh cells, andELISPOT assays. B) The inguinal lymph nodes were collected from immunized mice on day 28, and the GC B and Tfh cells. As demonstrated, all constructs showed the potential to significantly increase the percentage of GC B and Tfh cells when compared to negative control mice. Among the vaccine groups, the Nmut group differed significantly from the other three, but no significant differences among the vaccine groups in GC B cells were observed. A significantly higher percentage of antigen-specific GC B cells was detected in the vaccine groups compared to the negative control. No significant differences were observed among the vaccine groups. C) To quantify antigen-specific T cell responses, splenocytes were stimulated for 24 hours with 10 pg / mL of nucleocapsid protein and analyzed via ELISPOT for IFN-y and IL-4 secretion. For IFN-gamma, all four vaccine groups showed significantly higher levels than the negative control, but the nucleocapsid and Nmut groups showed significantly higher levels than GP38 and GP38Aglyc. On the other hand, GP38 and GP38Aglyc had greater potential to stimulate a significant level of IL-4 than nucleocapsid, Nmut, and the negative control. D) Inhouse ELISA was conducted to test the levels of antibody on day 28. One mouse in nucleocapsid and 3 in Nmut out of 8 showed a positive signal in a 1 :50 dilution. On the other hand, all the samples in the GP38 and GP38Aglyc groups were positive at 1 :8000 dilution. No significant differences among the vaccine groups were noted. Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparisons test. Significant differences are shown as p-values.

[0059] FIG. 36A-36G - shows immunogenicity of CleanCap M6 capped nucleocapsid, Nmut, GP38, and GP38Aglyc mRNAs with CCHFV UTR region in C57BL / 6 mice model in a booster regime. A) The same immunization schedule was used using CleanCap M6 mRNA / LNPs (nucleocapsid, Nmut, GP38 and GP38Aglyc). Blood, lymph node, and spleen were collected on day 28 for downstream immunological analyses, including ELISA, DC cells, and ELISPOT assays. B) The total CD4+ and CD8+ cells were analyzed in the collected splenocytes. As demonstrated, all four vaccine candidates showed significantly higher percentages of cells than the negative control group. No significant differences were observed among the vaccine groups, except for a significant difference between GP38 and GP38Aglyc for CD8+ cells. C) Splenocytes were isolated and stimulated ex vivo with 10 pg / mL of recombinant nucleocapsid or GP38 protein for 5 hours, followed by ICS for IFN-y, IL-2, and TNF-a in CD8+T cells. The nucleocapsid group showed a significantly higher level of IFN- gamma than the negative control, but no significant differences were observed among thevaccine groups. For IL-2, we observed the same pattern as IFN-gamma. On the other hand, both nucleocapsid and Nmut showed a significant level of TNF-alpha when compared to the negative control. D) In parallel, CD4+T cell responses were analyzed for IFN-y, IL-2, and TNF-a production. All vaccine constructs induced substantial cytokine expression compared to the control. However, only GP38Aglyc showed significantly higher levels of IL-2 and TNF- alpha than the negative control. E) In the ELISPOT assay, nucleocapsid and Nmut showed a significantly high level of IFN-gamma when compared to GP38, GP38Aglyc, and the negative control. Both GP38 and GP38Aglyc were unable to stimulate this response. In addition, all four vaccine candidates failed to elicit IL-4 responses in the ELISPOT after 24 hours of stimulation. F) In the ELISA assay, we detected only 3 positive samples for Nmut and none for nucleocapsid at a 1 :400 dilution. On the other hand, both GP38 and GP38Aglyc groups showed positivity in a 1 :8000 dilution. G) Lymph nodes were collected and stained to detect the pDC and cCDl+ population in the mice. As shown, only the GP38 and nucleocapsid groups showed significantly higher levels in pDC and cCDl, respectively, compared with the negative control group. Statistical analysis was performed using one-way ANOVA, followed by Sidak’s multiple comparisons test. Significant differences are shown as p-values.

[0060] FIG. 37 shows a schematic overview of a modular design framework for mRNA- lipid nanoparticle (LNP) vaccine candidates against Crimean-Congo hemorrhagic fever virus (CCHFV). The figure illustrates the rational design strategy integrating antigen selection, mRNA engineering, and LNP formulation. The CCHFV antigens evaluated include nucleocapsid (N), a mutant form of nucleocapsid (Nmut), glycoprotein 85 (GP85), and a modified GP85 variant lacking two N-glycosylation sites (GP38Aglyc). The mRNA constructs are optimized through regulatory element engineering, including segmented poly(A) tails (110 bp), distinct untranslated regions (UTRs), alternative 5' cap structures (Cap 0 or Cap 1), and incorporation of modified nucleotides such as N1-methylpseudouridine and 5-methylcytidine. The mRNAs are encapsulated within LNPs comprising a novel ionizable lipid (BP- 104) and helper lipids, providing efficient mRNA delivery and antigen expression. The modular platform enables systematic evaluation of sequence and formulation parameters that influence mRNA translation, innate immune sensing, and adaptive immune responses.

[0061] FIG. 38A-38F show a comparative analysis of ionizable lipids in mRNA-LNP formulations encoding a CCHFV mutant nucleocapsid (Nmut) for in vitro assessment of innate and adaptive immune stimulation. A) A novel ionizable lipid (BP- 104) was selected andcompared in its potential to stimulate immunity with two ionizable lipids, SM-102 and ALC- 0315. The pictures of chemical structures of these ionizable lipids were obtained from BROADPHARM Company (broadpharm.com). B) LNP composition and formulation. C) Formulations of LNPs generated by three different ionizable lipids were given at different doses to IFN-a / p Reporter HEK 293 Cells, and the IFN-I fold change relative to the negative control (1 *PBS) was documented. Empty LNPs (iLNP-BP104, iLNP-SM102, and iLNP-ALC- 0315) were also included in the assay. All mRNA / LNP constructs stimulated IFN-I responses after 24 hours as measured by SEAP activity. In addition, iLNPs triggered this response, showing their adjuvant potential. These constructs were also tested in THPl-Dual cells to evaluate their potential to stimulate innate immunity by activating the NF-KB pathway, and all mRNA / LNPs and iLNPs activated the pathway with no significant differences among groups. All three constructs also stimulated IL-6 production in HEKBlue cells, with no significant differences among the LNPs. D) In the antigen presentation experiment, 24-hour antigen- pulsed dendritic cells (Nmut-BP104, Nmut-SM102, and Nmut-ALC0315) were co-cultured with naive CD4+T cells from the spleens of C57BL / 6 mice for 4 days, and E) intracellular cytokines were measured after 5 hours of treatment with Brefeldin A. The Nmut-ALC0315 construct significantly enhanced CD4+cell activation, as evidenced by elevated levels of IFN- y, TNF-a, and IL- 17 A. F) Supernatants were collected to measure 13 antiviral cytokines and chemokines; no significant differences were observed between groups, except for MCP-1. Statistical analysis was performed using one-way ANOVA followed by post hoc Tukey’s multiple comparisons test, and significant differences are shown as p-values.

[0062] FIG. 39A-39G - shows the effect of ARCA capping on the expression and immunogenicity of CCHFV nucleocapsid mRNA-LNP in vitro and in vivo. A) ARCA-capped mRNAs were generated to express nucleocapsid by incorporating three different untranslated regions (UTRs), including UTR from the S segment of CCHFV (CCHFV-N UTR), an optimized UTR (T UTR), and the UTR region of the ACTA-1 gene (ACTA-1 UTR). These UTRs exhibit different levels of complexity, as shown by their secondary RNA structures generated with QIAGEN CLC Main Workbench 21.0. B) Gel electrophoresis of generated ARCA-capped mRNAs after purification using the Monarch RNA purification kit. C) Particle size and poly dispersity index (PDI). All data are presented as mean ± standard deviation, with n = 3 replicates. D) In this experiment, 8-10-week-old female C57BL / 6 mice were immunized with 10 pg of mRNA / LNPs at day 0 and day 14, and blood and spleens were collected toperform ELISA and intracellular cytokine staining. E) The splenocytes from immunized mice were collected and stimulated for 24 hours with 10 pg / ml of nucleocapsid to perform ELISPOT. All three vaccine candidates significantly increased IL-2 secretion compared to the negative control mice. On the other hand, significant differences were observed between the CCHFV and ACTA-1 UTRs and the T-UTR and negative control groups. F) The splenocytes were isolated from immunized mice on day 28 and stimulated for 5 hours with 10 pg / ml of nucleocapsid and then stained for intracellular cytokines of IFN-y, IL-2, and TNF-a in CD8+cells. As demonstrated, the T-UTR construct significantly stimulated the production of all these cytokines compared to the negative control. However, no significant differences were documented between the three vaccine candidates. G) Intracellular cytokines, including IFN- y, IL-2, IL-4, TNF-a, and IL-17A, were also measured in CD4+cells. The ACTA-l-UTR construct showed significantly higher levels of all five cytokines than the other vaccine and negative control groups. Interestingly, CCHFV- and T-UTR groups showed similar levels of these cytokines in CD4+cells. H) ELISA analysis of total IgG antibody responses against nucleocapsid protein in BALB / c mice immunized with ARCA-capped nucleocapsid and Nmut mRNA / LNPs. Positive antibody responses were detected in three of four mice in both vaccine groups at a 1 :50 dilution. Statistical analysis was performed using one-way ANOVA followed by post-hoc Tukey’s multiple comparisons test. Significant differences are shown as p-values.

[0063] FIG. 40A-40B - shows size exclusion chromatography of recombinant proteins. A) Size exclusion chromatography of nucleocapsid protein collected following Ni-NTA gravity purification from Rosetta bacteria, with Coomassie blue staining of corresponding fractions showing a pure nucleocapsid band at peak 6. B) Size exclusion chromatography of recombinant GP38 collected from ExpiCHO cell supernatant and purified using Strep-Tactin beads, with Coomassie blue staining of corresponding fractions showing a pure GP38 band at peak 6. For both experiments, a Superdex 75 10 / 300 GL column from Cytiva was used for final purification.

[0064] FIG. 41A-41G - shows the immunogenicity of AG-capped nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc) mRNAs containing CCHFV untranslated regions (UTRs) in a C57BL / 6 mouse model using a booster immunization regime. A) Structures of AG-capped mRNAs expressing nucleocapsid, mutant nucleocapsid (Nmut), GP85, and GP85 (GP38Aglyc) with UTRs derived from the S and M segments of CCHFV. B) Gel electrophoresis of AG- capped mRNAs following purification. C) Particle size and poly dispersity index (PDI) ofmRNA / LNP formulations, with data represented as mean ± standard deviation from three replicates. D) The immunization schedule for AG-capped mRNA / LNPs (nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc)) is shown. Blood, lymph nodes, and spleens were collected on day 28 for downstream analyses, including ELISA, germinal center (GC) B and T follicular helper (Tfh) cell quantification, and ELISPOT assays. E) Antigen-specific T cell responses were evaluated by stimulating splenocytes for 24 hours with 10 pg / mL of nucleocapsid or GP85 protein and performing ELISPOT for IFN-y and IL-4 secretion. The N and Nmut groups exhibited significantly higher IFN-y spot counts compared to GP85, GP85 (GP38Aglyc), and negative control, whereas GP85 and GP85 (GP38Aglyc) induced significantly higher IL-4 responses compared to N, Nmut, and negative control. F) GC B and Tfh responses were analyzed from inguinal lymph nodes collected on day 28. Nmut significantly increased the percentage of GC B and Tfh cells relative to negative control mice, and N also showed elevated GC B cell levels. All vaccine groups exhibited significantly higher frequencies of antigenspecific GC B cells than the negative control group, with no significant differences among vaccine groups. G) In-house ELISA analysis on day 28 showed two of eight Nmut-immunized mice with positive antibody signals, while all samples from GP85 and GP85 (GP38Aglyc) groups were significantly positive compared to the negative control. No significant differences were observed between GP85 and GP85 (GP38Aglyc) groups. Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple-comparison test, and significant differences are indicated by p-values.

[0065] FIG. 42A-42K - shows the immunogenicity of CleanCap M6-capped nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc) mRNAs containing CCHFV untranslated regions (UTRs) in a C57BL / 6 mouse model using a booster immunization regime. A) The structure of Cleancap M6-capped mRNAs with UTRs derived from the S and M segments of CCHFV. B) Gel electrophoresis of CleanCap M6-capped mRNAs following purification. C) Particle size and poly dispersity index (PDI) of mRNA / LNP formulations, with all data represented as mean ± standard deviation from three replicates. D) Cell proliferation assays were performed in HEK293T cells 24 hours after treatment with varying concentrations of CleanCap M6 mRNA / LNPs (nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc)) using the CCK-8 assay. Nmut mRNA / LNP showed a proliferation rate comparable to the negative control (l x PBS), whereas nucleocapsid exhibited the highest cytotoxicity. E) The generated CleanCap M6 mRNA / LNPs exhibited pKa values of 6.94 (nucleocapsid), 6.82 (Nmut), 6.73(GP85), and 6.62 (GP85 (GP38Aglyc)). F) These mRNAs were transfected into HepG2, C2C12, and HEK293T cells, and protein expression was confirmed by western blot showing expected bands at 54 kDa for nucleocapsid / Nmut and 38 kDa for GP38 / GP38Aglyc. G) The immunization schedule for CleanCap M6 mRNA / LNPs (nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc)) is shown. Blood, lymph nodes, and spleens were collected on day 35 for downstream analyses, including ELISA, dendritic cell activation, and ELISPOT assays. H) Splenocytes were stimulated for 24 hours with 10 pg / mL of nucleocapsid and GP38 proteins, followed by ELISPOT assays for IFN-y and IL-4 secretion. N and Nmut vaccine groups exhibited significantly higher IFN-y responses than GP85, GP85 (GP38Aglyc), and the negative control, while all four vaccine groups failed to induce IL-4 responses significantly. I) ELISA analyses on day 35 revealed three positive samples among six Nmut-immunized mice and none for nucleocapsid, whereas GP85 and GP85 (GP38Aglyc) groups displayed significantly higher antibody levels relative to the negative control, with no significant difference between these two groups. J) Lymph nodes were collected and stained to assess plasmacytoid dendritic cell (pDC) and cDCl+populations; GP85 and nucleocapsid groups showed significantly higher pDC and cDCl+levels, respectively, compared to the negative control. K) The gating strategy to evaluate intracellular cytokines in CD4+and CD8+T cells is described. Splenocytes were isolated and stimulated ex vivo with 10 pg / mL of recombinant nucleocapsid or GP38 proteins for five hours, followed by intracellular cytokine staining for IFN-y, IL-2, and TNF-a. All vaccine groups showed significantly elevated total CD4+and CD8+T cells compared with the negative control, except for GP85 (GP38Aglyc). Among vaccine groups, nucleocapsid induced the highest CD8+responses for IFN-y, IL-2, and TNF- a, while GP85 (GP38Aglyc) significantly increased IL-2 and TNF-a production in CD4+cells compared to the negative control. No significant differences were observed among vaccine groups. Statistical analysis was performed using one-way ANOVA followed by Sidak’s multiple-comparison test, and p-values indicate significant differences.

[0066] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0067] The present disclosure provides novel lipid particle (LP) formulations that address fundamental limitations in mRNA vaccine delivery through systematic molecular andnanomaterial engineering. The disclosed LPs comprise optimized combinations of ionizable lipids, neutral lipids, steroids, and polymer-conjugated lipids that collectively enhance mRNA encapsulation, cellular delivery, and immune activation while minimizing adverse reactogenicity.

[0068] The LPs encapsulate RNA molecules comprising naturally occurring ribonucleotides, chemically modified nucleotides, or combinations thereof, collectively encoding one or more therapeutic proteins. The encapsulated RNA can include modified nucleotides such as N1 -methylpseudouridine and 5-methylcytidine that enhance stability and reduce unwanted innate immune activation while maintaining translation efficiency. The RNA constructs incorporate optimized regulatory elements, including pathogen-derived UTRs, synthetic cap structures, and segmented poly(A) tails that collectively influence translation kinetics and immune recognition patterns.

[0069] A key innovation lies in the systematic engineering framework that enables rational optimization of multiple interconnected parameters. The disclosed approach recognizes that molecular components, including UTR selection, cap structure, and nucleoside modifications, interact with nanomaterial properties such as ionizable lipid chemistry to determine overall vaccine performance. This integrated optimization strategy enables the development of formulations tailored to specific immune outcome requirements while maintaining manufacturing scalability and clinical feasibility.

[0070] The lipid compositions feature novel ionizable lipids, including the specifically disclosed compound heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2- hydroxyethyl)amino)heptanoate (BP- 104), that provide enhanced mRNA delivery with favorable safety profiles. These ionizable lipids enable pH-dependent membrane fusion and endosomal escape while minimizing systemic toxicity. The formulations incorporate precise molar ratios of helper lipids, including neutral phospholipids, cholesterol, and PEGylated lipids, which collectively stabilize particle formation and optimize biodistribution.

[0071] The disclosed RNA vaccines demonstrate the ability to trigger balanced immune responses encompassing both cellular and humoral immunity. By carefully optimizing molecular and nanomaterial parameters, the formulations can be tailored to favor specific immune outcomes, such as Th 1 -polarized cellular responses or robust antibody production, depending on pathogen-specific requirements. This flexibility enables rational vaccine designfor diverse infectious disease targets while maintaining platform consistency and regulatory pathway efficiency.

[0072] Importantly, the benefits of these LP formulations are not dependent on specific RNA sequences, enabling rapid adaptation to encode antigens from various pathogens. This sequence-agnostic approach facilitates rapid deployment during epidemic or pandemic scenarios while maintaining optimized delivery and immune activation. The platform's modularity supports the development of monovalent vaccines targeting individual pathogens or multivalent formulations addressing multiple threats simultaneously.

[0073] In one aspect, embodiments disclosed herein address unmet needs in vaccine development for high-consequence pathogens exemplified by CCHFV, where case fatality rates approach 40% and no licensed countermeasures exist. However, the systematic engineering principles and optimized formulations described herein have broad applicability across diverse viral, bacterial, and oncological targets, establishing a versatile platform for next-generation therapeutic development.

[0074] By integrating rational molecular design with advanced nanomaterial engineering, the disclosed LP formulations overcome the fundamental limitations of current mRNA vaccine platforms. The systematic approach enables predictable optimization of key performance parameters, including translation efficiency, immune activation balance, and safety profiles, while maintaining the inherent advantages of mRNA technology, including rapid development timelines, manufacturing scalability, and adaptive immune stimulation. This comprehensive platform establishes a foundation for addressing both current and emerging global health challenges through rationally engineered therapeutic interventions.LIPID NANOPARTICLES

[0075] Disclosed herein are compounds that can form lipid particles with other lipid components, including polymer-conjugated lipids, cationic lipids, neutral lipids, and / or sterols. These lipid particles can form complexes with oligonucleotides. Without being bound by theory, these lipid particles are believed to protect oligonucleotides from serum degradation and facilitate their efficient delivery to cells in vitro and in vivo.

[0076] In an example embodiment, the compound is a compound of Formula I:or a pharmaceutically acceptable salt thereof, wherein A1and A2are each independently, optionally substituted alkylene (e.g., Ci-C 24 alkylene, C1-C15 alkylene, C1-C12 alkylene).

[0077] In an example embodiment, A1and A2are each independently optionally substituted with one to twelve carbon atoms (C1-C12 alkylene).

[0078] In an example embodiment, A1and A2are each independently optionally substituted methylene (Ci-alkylene), optionally substituted ethylene (C2-alkylene), optionally substituted propylene (Cs-alkylene), optionally substituted butylene (C4-alkylene), optionally substituted pentylene (Cs-alkylene), optionally substituted hexylene (Ce-alkylene), optionally substituted heptylene (C?-alkylene), optionally substituted octylene (Cs-alkylene), optionally substituted nonylene (C9-alkylene), optionally substituted decylene (Cio-alkylene), optionally substituted undecylene (Cn -alkylene), or optionally substituted dodecylene (Cn-alkylene).

[0079] In an example embodiment, A1and A2are each independently unsubstituted methylene (Ci-alkylene), unsubstituted ethylene (C2-alkylene), unsubstituted propylene (Cs- alkylene), unsubstituted butylene (C4-alkylene), unsubstituted pentylene (Cs-alkylene), unsubstituted hexylene (Ce-alkylene), unsubstituted heptylene (C?-alkylene), unsubstituted octylene (Cs-alkylene), unsubstituted nonylene (C9-alkylene), unsubstituted decylene (C10- alkylene), unsubstituted undecylene (Cn-alkylene), or unsubstituted dodecylene (Cn- alkylene).

[0080] In an example embodiment, A1and A2are each independently optionally substituted with a propylene (Cs-alkylene), hexylene (Ce-alkylene), or nonylene (Cg-alkylene) group.

[0081] In an example embodiment, A1and A2are each independently unsubstituted propylene (Cs-alkylene), unsubstituted hexylene (Ce-alkylene), or unsubstituted nonylene (C9- alkylene).

[0082] In an example embodiment, A1is optionally substituted hexylene (Ce-alkylene).

[0083] In an example embodiment, A1is unsubstituted hexylene (Ce-alkylene).

[0084] In an example embodiment, A1is optionally substituted nonylene (C9-alkylene).

[0085] In an example embodiment, A1is unsubstituted nonylene (C9-alkylene).

[0086] In an example embodiment, A2is optionally substituted hexylene (Ce-alkylene).

[0087] In an example embodiment, A2is unsubstituted hexylene (Ce-alkylene).

[0088] In an example embodiment, A2is optionally substituted nonylene (Cg-alkylene).

[0089] In an example embodiment, A2is unsubstituted nonylene (Ce-alkylene).

[0090] In an example embodiment, the compound of Formula I is heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2-hydroxyethyl)amino)heptanoate.or a pharmaceutically acceptable salt thereof. The compound of Formula I is referred to herein as Compound 1, which is interchangeably referred to as BP-104, representing the novel ionizable lipid employed in the lipid nanoparticle formulations described herein, including in Example 1.

[0091] In an example embodiment, the compound of Formula I is present in a ratio of from about 20 mol % to about 70 or 75 mol % or from about 45 to about 65 mol% or about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol % of the total lipid present in the LP. In an example embodiment, the LPs comprise from about 25 mol% to about 75 mol% on a molar basis of cationic lipid, e.g., from about 20 mol% to about 70 mol%, from about 35 mol% to about 65 mol%, from about 45 mol% to about 65 mol%, about 60 mol%, about 55 mol%, about 50%, or about 40% on a molar basis (based upon 100% total moles of lipid in the lipid particle). In an example embodiment, the ratio of cationic lipid to nucleic acid is about 3 to about 15, from about 5 to about 13 or 7 to 11.

[0092] The amount of the cationic lipid may be selected based on the amount of the nucleic acid cargo. In one embodiment, these amounts are selected to yield an N / P ratio for the particle(s) or the composition in the range of about 0.1 to about 20. In this context, the N / P ratio is defined as the mole ratio of the nitrogen atoms (“N”) of the basic nitrogen-containing groups of the lipid to the phosphate groups (“P”) of the RNA, which is used as cargo. The N / Pratio may be calculated on the basis that, for example, 1 pg RNA typically contains about three nmol phosphate residues, provided that the RNA exhibits a statistical distribution of bases. The “N”-value of the lipid may be calculated based on its molecular weight and the relative content of permanently cationic and — if present — cationizable groups.

[0093] In an example embodiment, the LP comprises one or more additional lipids that stabilize the formation of particles during their formation.

[0094] In an example embodiment, a non-cationic may be used. The non-cationic lipid can be a neutral lipid, an anionic lipid, or an amphipathic lipid. Neutral lipids, when present, can be any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at physiological pH. Such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. The selection of neutral lipids for use in the particles described herein is generally guided by consideration of, e.g., LP size and stability of the LP in the bloodstream. Preferably, the neutral lipid has two acyl groups (e.g., diacylphosphatidylcholine and di acy Iphosphati dy 1 ethanol amine) .

[0095] In an example embodiment, the neutral lipids contain saturated fatty acids with carbon chain lengths of CIO to C20. In other embodiments, neutral lipids with mono or diunsaturated fatty acids with carbon chain lengths in CIO to C20 are used. Additionally, neutral lipids having mixtures of saturated and unsaturated fatty acid chains can be used.

[0096] Suitable neutral lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyl oleoyl phosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-I-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), dimyristoyl phosphatidylcholine (DMPC), distearoyl-phosphatidyl-ethanolamine (DSPE), SM, 16-0- monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, I-stearoyl-2-oleoyl- phosphatidy ethanolamine (SOPE), cholesterol, or a mixture thereof. Anionic lipids suitable for use in LPs include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine,lysylphosphatidylglycerol, and other anionic modifying groups joined to neutral lipids. In one embodiment, the neutral lipid is l,2-distearoyl-sn-glycero-3 phosphocholine (DSPC). In an example embodiment, the LPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM. In various embodiments, the molar ratio of the cationic lipid to the neutral lipid ranges from about 2: 1 to about 8: 1.

[0097] Amphipathic lipids refer to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and betaacyloxy acids, can also be used.

[0098] Amphipathic lipids refer to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Such compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatdylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds, such as sphingolipids, glycosphingolipid families, diacylglycerols, and betaacyloxy acids, can also be used.

[0099] In an example embodiment, the non-cationic lipid is present in a ratio of from about 5 mol % to about 90 mol %, about 5 mol % to about 10 mol %, about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or about 90 mol % of the total lipid present in the LP.

[0100] In an example embodiment, LPs comprise from about 0% to about 15 or about 45% on a molar basis of neutral lipid, e.g., from about 3 to about 12% or from about 5 to about 10%. For instance, LPs may include about 15%, about 10%, about 7.5%, or about 7.1% ofneutral lipid on a molar basis (based upon 100% total moles of lipid in the LP). In an example embodiment, a sterol may be used. In an example embodiment, the sterol is cholesterol.

[0101] In an example embodiment, an aggregation-reducing agent may be employed. The aggregation-reducing agent can be a lipid capable of reducing aggregation.

[0102] Examples of such lipids include but are not limited to, polyethylene glycol (PEG)- modified lipids, monosialoganglioside Gml, and polyamide oligomers (PAO) such as those described in U.S. Pat. No. 6,320,017, which is incorporated by reference in its entirety. Other compounds with uncharged, hydrophilic, steric-b airier moieties, which prevent aggregation during formulation, like PEG, Gml, or ATTA, can also be coupled to lipids. ATTA-lipids are described, e.g., in U.S. Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., in U.S. Pat. Nos. 5,820,873, 5,534,499, 5,885,613, US20150376115A1, and WO2015 / 199952, each of which is incorporated by reference in its entirety.

[0103] The aggregation reducing agent may be, for example, selected from polyethylene glycol (PEG)-lipid , including, without limitation, a PEG-diacylglycerol (DAG), aPEG-dialkyl glycerol, a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof (such as PEG-Cerl4 or PEG-Cer20). The PEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), a PEG- dipalmityloxypropyl (Cl 6), or a PEG-distearyloxypropyl (Cl 8). Other pegylated-lipids include, but are not limited to, polyethylene glycol-didimyristoyl glycerol (C14-PEG or PEG- C14, where PEG has an average molecular weight of 2000 Da) (PEG-DMG); (R)-2,3- bis(octadecyloxy)propyl-l-(methoxy poly(ethylene glycol)2000) propyl carbamate) (PEG- DSG); PEG-carbamoyl-l,2-dimyristyloxypropylamine, in which PEG has an average molecular weight of 2000 Da (PEG-cDMA); N-Acetylgalactosamine-((R)-2,3- bis(octadecyloxy)propyl-l-(methoxy polyethylene glycol)2000) propyl carbamate) (GalNAc- PEG-DSG); mPEG (mw2000)-diastearoylphosphatidyl-ethanolamine (PEG-DSPE); and polyethylene glycol-dipalmitoylglycerol (PEG-DPG).

[0104] In an example embodiment, the aggregation-reducing agent is PEG-DMG 2000. In other embodiments, it is PEG-c-DMA.

[0105] In an example embodiment, the molar ratio of the cationic lipid to the PEGylated lipid ranges from about 100:1 to about 25: 1.

[0106] In an example embodiment, the LP additionally comprises one or more lipids that stabilize particle formation (e.g., a neutral lipid and / or one or more steroid or steroid analogs).

[0107] In preferred embodiments of the second aspect, the artificial RNA of the first aspect and, optionally, the further artificial RNA of the second aspect, is complexed with one or more lipids, thereby forming lipid particles (LP), wherein the LP additionally comprises one or more neutral lipids and / or one or more steroid or steroid analogs.

[0108] Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.Oligonucleotide Payload

[0109] In an example embodiment, the lipid particles described herein comprise an oligonucleotide cargo. The oligonucleotide may be any therapeutic or prophylactic nucleic acid suitable for delivery via lipid particle encapsulation. In an example embodiment, the oligonucleotide is an antisense oligonucleotide. In an example embodiment, the oligonucleotide is messenger RNA (mRNA). In an example embodiment, the oligonucleotide is both antisense and mRNA.

[0110] The oligonucleotide cargo is encapsulated within the lipid particle, protected from degradation, and delivered to target cells where it can exert its therapeutic or prophylactic effect. As described above, lipid particles typically exhibit high encapsulation efficiency (e.g., at least 90%) and appropriate N / P ratios, ensuring effective oligonucleotide loading and cellular delivery. mRNA Structure and Components[OHl] When the oligonucleotide comprises mRNA, the mRNA may include various structural and functional elements that enhance its stability, translation efficiency, and immunogenicity profile. In an example embodiment, the mRNA comprises a coding region that encodes at least one polypeptide or protein of interest. In an example embodiment, the mRNA comprises a coding region that encodes at least one antigenic polypeptide or protein. In an example embodiment, the antigenic polypeptide or protein comprises a sequence modification relative to a wild-type or naturally occurring sequence. In an example embodiment, the sequence modification is selected from modifications to the nucleocapsid protein, the GP38 protein, or their mutants. 1

[0112] In an example embodiment, beyond the coding region, the mRNA additionally comprises one or more structural elements that enhance mRNA function. These structural elements may include, without limitation, a 5'-untranslated region (5'-UTR) structure, a poly(A) sequence, a poly(C) sequence, a 3 '-untranslated region (3'-UTR) structure, a 5' cap structure, or any combination thereof.5' Cap Structure

[0113] In an example embodiment, the mRNA comprises a 5' cap structure. The 5' cap is a modified guanosine nucleotide added to the 5' end of the mRNA that protects the mRNA from degradation and enhances translation efficiency by facilitating ribosome binding and preventing recognition by innate immune sensors.

[0114] In an example embodiment, the 5' cap structure comprises a 7-methylguanosine (m7G) moiety linked to the first transcribed nucleotide via a S' -S' triphosphate bridge. The cap structure may be added enzymatically following transcription or may be incorporated co- transcriptionally using modified cap analogs.

[0115] In an example embodiment, the 5' cap structure comprises a cap analog that is incorporated during in vitro transcription. Cap analogs may include anti-reverse cap analogs (ARCAs) that contain chemical modifications preventing incorporation in the reverse orientation, thereby ensuring proper cap orientation and enhanced translation efficiency. In an example embodiment, the cap analog is an Anti-Reverse Cap Analog (ARCA), which may comprise 3 '-O-m ethylation of the 7-methylguanosine moiety.

[0116] In an example embodiment, the 5' cap structure comprises a dinucleotide cap structure. A dinucleotide cap may comprise a 7-methylguanosine linked to an adenosine, forming a m7G-5'-ppp-5'-A structure. In an example embodiment, the dinucleotide cap is an adenosine-guanosine (AG) cap.

[0117] In an example embodiment, the 5' cap structure comprises a trinucleotide cap structure. In an example embodiment, the cap structure further comprises one or more methylated nucleotides, including N6-methyladenosine (m6A) or 2'-O-methylation of ribose sugars at the first or second transcribed nucleotide positions. Such methylation patterns may mimic naturally occurring Cap 1 or Cap 2 structures found in eukaryotic mRNAs. In an example embodiment, the cap structure comprises a 7-methylguanosine linked to an N6- methyladenosine (m7G-5'-ppp-5'-m6A structure), optionally with additional 2'-O-methylation. In an example embodiment, the 5' cap comprises a trinucleotide analog that produces abase-modified Cap 1 structure, m7G-ppp-m6Am-N, in which the first transcribed nucleotide is N6-methyladenosine and is 2'-O-methylated, such as CleanCap M6 (TriLink Biotechnologies).

[0118] Other suitable cap structures known in the art may also be employed, including enzymatically added caps using vaccinia virus capping enzyme or other capping systems.5 '-Untranslated Region (5'- UTR).

[0119] In an example embodiment, the mRNA comprises a 5'-UTR structure positioned between the 5' cap and the start codon of the coding region. The 5'-UTR is a regulatory sequence that can significantly influence translation efficiency, mRNA stability, and localization. The 5'-UTRmay affect ribosome recruitment, scanning efficiency, and translation initiation at the start codon. In an example embodiment, the 5'-UTR has a length of 10 to 500 nucleotides, 20 to 400 nucleotides, 30 to 300 nucleotides, 40 to 200 nucleotides, or 50 to 150 nucleotides. The optimal length may be selected based on the specific coding sequence and desired expression characteristics.

[0120] In an example embodiment, the 5'-UTR is selected or designed to minimize secondary structure formation that could impede ribosome scanning. Highly structured 5'- UTRs with stable stem-loops or extensive base pairing can reduce translation efficiency by creating barriers to ribosome scanning from the 5' cap to the start codon. In an example embodiment, the 5'-UTR has a minimum free energy greater than -30 kcal / mol, -25 kcal / mol, or -20 kcal / mol to facilitate efficient ribosome scanning.

[0121] In an example embodiment, the 5'-UTR comprises or is positioned adjacent to a Kozak consensus sequence surrounding the start codon. The Kozak sequence provides an optimal context for translation initiation. In an example embodiment, the Kozak sequence comprises the consensus (gcc)gccRccAUGG, where R represents a purine (A or G), and the start codon AUG is shown in uppercase.

[0122] In an example embodiment, the 5'-UTR is derived from a viral sequence, a mammalian gene, or a synthetic sequence optimized for translation efficiency. In an example embodiment, the 5'-UTR structure is selected from CCHFV-UTR, T-UTR, and ACTA-UTR. The CCHFV-UTR is derived from the 5' untranslated region of the S segment of Crimean- Congo hemorrhagic fever virus. The T-UTR may be derived from human alpha-globin or betaglobin genes, or from other highly expressed genes known to confer efficient translation. The ACTA-UTR corresponds to the 5' UTR region of the ACTA-1 gene encoding skeletal musclealpha-actin, the predominant actin isoform in adult skeletal muscle, which is known for high translational efficiency in muscle tissue.

[0123] In an example embodiment, the 5'-UTR is selected to enhance tissue-specific or cell-type-specific translation. Different 5'-UTR sequences may exhibit varying translation efficiencies depending on the cellular environment, abundance of translation factors, and the presence of specific RNA-binding proteins. In an example embodiment, the 5'-UTR is selected to enhance translation in immune cells, muscle cells, hepatocytes, or other target cell types. These and other UTR sequences may be selected based on their ability to enhance translation in specific cell types or contexts, or to achieve desired expression levels of the encoded protein. 3 '-Untranslated Region (3'-UTR).

[0124] In an example embodiment, the mRNA comprises a 3'-UTR structure positioned between the stop codon and downstream elements such as the poly(A) sequence or poly(C) sequence. The 3'-UTR is a regulatory region that influences mRNA stability, localization, translation efficiency, and post-transcriptional regulation. The 3 '-UTR may contain binding sites for RNA-binding proteins, microRNAs, and other regulatory factors that modulate mRNA fate and function.

[0125] In an example embodiment, the 3 '-UTR has a length of 10 to 1000 nucleotides, or 20 to 800 nucleotides, or 30 to 600 nucleotides, or 50 to 500 nucleotides, or 100 to 400 nucleotides. The optimal length may be selected based on the desired expression kinetics and mRNA half-life.

[0126] In an example embodiment, the 3 '-UTR influences mRNA stability by providing binding sites for stabilizing or destabilizing RNA-binding proteins. Certain 3'-UTRs may contain AU-rich elements (AREs) that promote rapid mRNA decay, while others lack such elements and confer enhanced stability. In an example embodiment, the 3'-UTR is selected to enhance mRNA stability and prolong the duration of protein expression. In an example embodiment, the 3'-UTR is selected to provide shorter mRNA half-life and more transient protein expression.

[0127] In an example embodiment, the 3 '-UTR contributes to translational regulation through interactions with translation factors and through long-range interactions with the 5'- UTR. The poly(A)-binding proteins that bind to the poly(A) tail can interact with translation initiation factors bound to the 5' cap, circularizing the mRNA and enhancing translation efficiency. The 3'-UTR structure and sequence can modulate these interactions.

[0128] In an example embodiment, the 3'-UTR is derived from a viral sequence, a mammalian gene known for stable and efficient expression, or a synthetic sequence optimized for desired expression characteristics. In an example embodiment, the 3'-UTR structure is selected from CCHFV-UTR, T-UTR, and ACTA1-UTR. The CCHFV-UTR is derived from the 3' untranslated region of the S segment of Crimean-Congo hemorrhagic fever virus. The T- UTR may be derived from human alpha-globin or beta-globin genes, which are known to confer mRNA stability and efficient translation. The ACTA1-UTR corresponds to the 3' UTR region of the ACTA-1 gene encoding skeletal muscle alpha-actin, which provides stable expression in muscle tissue.

[0129] In an example embodiment, the 3'-UTR may comprise one or more regulatory elements including polyadenylation signals, downstream regulatory elements, or binding sites for specific RNA-binding proteins that modulate expression in particular cellular contexts. As with the 5'-UTR, 3'-UTR sequences may be selected to optimize mRNA performance in the intended application, including achieving desired expression levels, expression duration, or tissue-specific expression patterns.Poly(A) Sequence.

[0130] In an example embodiment, the mRNA comprises a poly(A) sequence (also referred to as a poly(A) tail or polyadenylate tail). The poly(A) sequence is typically located at the 3' end of the mRNA, downstream from the 3 '-UTR or other 3' regulatory elements. The poly(A) sequence consists of multiple consecutive adenosine monophosphates linked by standard phosphodiester bonds.

[0131] In an example embodiment, the poly(A) tail contains 10 to 300 adenosine monophosphates. In an example embodiment, the poly(A) tail contains 20 to 300, or 30 to 300, or 40 to 300, or 50 to 300, or 50 to 250, or 60 to 250, or 70 to 250, or 80 to 250, or 90 to 250, or 100 to 250, or 100 to 200, or 120 to 200, or 150 to 250 adenosine monophosphates. The optimal poly(A) tail length may be selected based on desired expression characteristics, with longer tails generally associated with enhanced stability and translation.

[0132] In an example embodiment, the poly(A) tail protects mRNA from enzymatic degradation in the cytoplasm by 3'-5' exonucleases. The poly(A) tail provides a buffer region that must be degraded before exonucleases can reach the coding sequence. In an example embodiment, the poly(A) tail aids in mRNA export from the nucleus to the cytoplasm by serving as a binding site for nuclear export factors. In an example embodiment, the poly(A) tailenhances translation by recruiting poly(A)-binding proteins (PABPs), which interact with translation initiation factors at the 5' cap, facilitating ribosome recruitment and improving translation efficiency.

[0133] In an example embodiment, the poly(A) sequence is a continuous, uninterrupted stretch of adenosine monophosphates. In an example embodiment, the poly(A) sequence is a segmented poly (A) tail comprising stretches of adenosine monophosphates interrupted by short linker sequences. Segmented poly(A) tails may comprise two or more adenosine-rich regions separated by linkers of 1 to 10 nucleotides that may contain guanosine, cytidine, or uridine residues. In an example embodiment, a segmented poly(A) tail comprises alternating blocks of 20-60 adenosines separated by linkers of 2-5 nucleotides.

[0134] In an example embodiment, the poly(A) sequence is encoded in a DNA template and transcribed during mRNA synthesis. In an example embodiment, the poly(A) sequence is added post-transcriptionally by enzymatic polyadenylation using poly(A) polymerase. The poly(A) tail addition method may be selected based on the mRNA production platform and the desired tail characteristics.

[0135] In an example embodiment, the length of the poly(A) tail is optimized to balance mRNA stability and translation efficiency with manufacturing considerations, as very long homopolymeric sequences can present challenges during synthesis and quality control.Poly(C) Sequence.

[0136] In an example embodiment, the mRNA comprises a poly(C) sequence (also referred to as a poly(C) tract or polycytidylate sequence). A poly(C) sequence comprises multiple consecutive cytidine monophosphates linked by standard phosphodiester bonds. The poly(C) sequence represents an additional structural element that can modulate mRNA stability, translation efficiency, and cellular localization.

[0137] In an example embodiment, the poly(C) sequence contains 10 to 200 cytidine monophosphates. In an example embodiment, the poly(C) sequence contains 10 to 200, or 15 to 200, or 20 to 200, or 20 to 150, or 25 to 150, or 30 to 150, or 30 to 100, or 40 to 100, or 50 to 100, or 30 to 80, or 40 to 80, or 50 to 80 cytidine monophosphates. The optimal poly(C) sequence length may be selected based on desired functional characteristics and compatibility with other mRNA structural elements.

[0138] The poly(C) sequence may be positioned at various locations within the mRNA construct. In an example embodiment, the poly(C) sequence is positioned between the 3'-UTRand the poly(A) sequence. In an example embodiment, the poly(C) sequence is positioned downstream of the poly(A) sequence, at the 3' terminus of the mRNA. In an example embodiment, the poly(C) sequence is positioned between the stop codon and the 3'-UTR. In an example embodiment, the poly(C) sequence is positioned between the 5' cap and the 5'-UTR. The position of the poly(C) sequence may be selected based on the desired functional impact and compatibility with other regulatory elements.

[0139] In an example embodiment, the poly(C) sequence can enhance mRNA stability by influencing secondary structure formation, affecting interactions with RNA-binding proteins, or modulating recognition by the cellular degradation machinery. Cytidine-rich sequences may form specific secondary structures or interact with poly(C)-binding proteins, thereby affecting mRNA half-life and localization. In an example embodiment, the poly(C) sequence enhances mRNA stability, thereby prolonging protein expression.

[0140] In an example embodiment, the poly(C) sequence may influence translation efficiency. The mechanism by which poly(C) sequences affect translation may involve alterations in mRNA circularization, effects on ribosome recruitment or processivity, or interactions with translation regulatory factors. In an example embodiment, the poly(C) sequence enhances translation initiation or elongation efficiency. In an example embodiment, the combination of a poly(C) sequence with a poly(A) sequence provides synergistic effects on mRNA stability and translation compared to either element alone.

[0141] In an example embodiment, the poly(C) sequence is a continuous, uninterrupted stretch of cytidine monophosphates. In an example embodiment, the poly(C) sequence comprises predominantly cytidine residues, with occasional interruptions (e.g., less than 10%, less than 5%, or less than 2%) by other nucleotides, such as uridine or adenosine. The degree of sequence purity may be selected based on desired functional characteristics.

[0142] In an example embodiment, the poly(C) sequence may interact with specific cellular poly(C)-binding proteins, including a-complex proteins (aCPs) or poly(C)-binding proteins (PCBPs), which are known to bind cytidine-rich RNA sequences and influence mRNA stability, localization, and translation. The presence of a poly(C) sequence may thereby provide a mechanism for modulating mRNA behavior through these protein-RNA interactions.

[0143] In an example embodiment, the mRNA comprises both a poly(C) sequence and a poly(A) sequence. When both sequences are present, they may be directly adjacent or separated by intervening sequences, such as linker sequences of 1 to 50 nucleotides, 1 to 30 nucleotides,1 to 20 nucleotides, or 1 to 10 nucleotides. The spatial relationship between the poly(C) and poly(A) sequences can be optimized to achieve desired expression characteristics.

[0144] In an example embodiment, the poly(C) sequence is selected or designed to avoid forming undesired secondary structures with other portions of the mRNA, such as the coding sequence or UTRs, which could impair translation or stability. Computational RNA structure prediction tools may be used to assess potential interactions and optimize poly(C) sequence placement and length.

[0145] In an example embodiment, the mRNA comprises two or more of these structural elements. In an example embodiment, the mRNA comprises a 5' cap, a 5'-UTR, a coding region, a 3'-UTR, and a poly(A) sequence. In an example embodiment, the mRNA comprises a 5' cap, a 5'-UTR, a coding region, a 3'-UTR, a poly(C) sequence, and a poly(A) sequence. Other combinations of structural elements are also contemplated and may be selected based on the specific requirements of the encoded protein and intended application.Chemically-Modified RNA

[0146] In an example embodiment, RNA (e.g., mRNA) vaccines of the present disclosure comprise at least one ribonucleic acid (RNA) oligonucleotide having an open reading frame encoding at least one virus antigenic polypeptide, wherein said RNA comprises at least one chemical modification.

[0147] The terms “chemical modification” and “chemically modified” refer to modification with respect to adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides in at least one of their position, pattern, percent or population. Generally, these terms do not refer to the ribonucleotide modifications in naturally occurring 5 '-terminal mRNA cap moi eties.

[0148] Modifications of oligonucleotides include, without limitation, those described herein and include, but are expressly not limited to, those modifications that comprise chemical modifications. Oligonucleotides (e.g., RNA, such as mRNA) may comprise naturally occurring, non-naturally-occurring modifications, or oligonucleotides may comprise a combination of naturally occurring and non-naturally-occurring modifications. Oligonucleotides may include any helpful modification, such as a sugar, a nucleobase, or an internucleoside linkage (e.g., to a linking phosphate, a phosphodiester linkage, or the phosphodiester backbone).

[0149] Concerning a polypeptide, “modification” refers to a modification relative to the canonical set of 20 amino acids. Polypeptides, as provided herein, are also considered “modified” if they contain amino acid substitutions, insertions, or a combination of substitutions and insertions.

[0150] In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) comprise various (more than one) different modifications. In an example embodiment, a particular oligonucleotide region contains one, two, or more (optionally different) nucleoside or nucleotide modifications. In an example embodiment, a modified RNA (e.g., a modified mRNA), introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified oligonucleotide. In an example embodiment, a modified RNA (e.g., a modified mRNA) introduced into a cell or organism may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response).

[0151] In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) comprise non-natural modified nucleotides introduced during synthesis or post-synthesis of the oligonucleotides to achieve desired functions or properties. The modifications may include internucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of an oligonucleotide may be chemically modified.

[0152] The present disclosure provides for modified nucleosides and nucleotides of a polynucleotide (e.g., RNA, such as mRNA). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may be synthesized by any useful method, such as chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Oligonucleotides may comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages may be standard phosphodiester linkages, in which case the oligonucleotides would comprise regions of nucleotides.

[0153] Modified nucleotide base pairing encompasses not only the standard adenosinethymine, adenosine-uracil, or guanosine-cytosine base pairs but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, whereinthe arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary nonstandard base structures, such as, for example, in those oligonucleotides having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil. Any base / sugar or linker combination may be incorporated into the present disclosure oligonucleotides.

[0154] Modifications of oligonucleotides (e.g., RNA, such as mRNA), including but not limited to chemical modification, that are useful in the compositions, vaccines, methods and synthetic processes of the present disclosure include, but are not limited to the following: 2- methylthio-N6-(cis-hydroxyisopentenyl)adenosine; 2-methylthio-N6-methyladenosine; 2- methylthio-N6-threonyl carbamoyladenosine; N6-glycinylcarbamoyladenosine; N6- isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladeno sine; l,2'-O- dimethyladenosine; 1 -methyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6 isopentenyladenosine; 2-methylthio-N6- hydroxynorvalyl carbamoyladenosine; 2'-O-methyladenosine; 2'-O-ribosyladenosine (phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine; N6,2'-O- dimethyladenosine; N6,2'-O-dimethyladenosine; N6,N6,2'-O-trimethyladenosine; N6,N6- dimethyladenosine; N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine; N6- methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine; 2-methylthio-N6- isopentenyladenosine; 7-deaza-adenosine; Nl-methyl-adenosine; N6,N6 (dimethyl)adenine; N6-cis-hydroxy-isopentenyl-adenosine; a-thio-adenosine; 2 (amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6 (isopentenyl)adenine; 2-(alkyl)adenine; 2- (aminoalkyl)adenine; 2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine; 2- (propyl)adenine; 2'-Amino-2'-deoxy-ATP; 2'-Azido-2'-deoxy-ATP; 2'-Deoxy-2'-a- aminoadenosine TP; 2'-Deoxy-2'-a-azidoadenosine TP; 6 (alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine; 7 (deaza)adenine; 8 (alkenyl)adenine; 8(alkynyl)adenine; 8 (amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine; 8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine; 8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine; 8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine; N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine; 1- Deazaadenosine TP; 2'Fluoro-N6-Bz-deoxyadenosine TP; 2'-OMe-2-Amino-ATP; TO- methyl-N6-Bz-deoxyadenosine TP; 2'-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP; 2-Amino-ATP; 2'-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP; 2'-b-Ethynyladenosine TP; 2-Bromoadenosine TP; 2'-b-Trifluoromethyladenosine TP; 2- Chloroadenosine TP; 2 '-Deoxy-2 ',2 '-difluoroadenosine TP; 2'-Deoxy-2'-a-mercaptoadenosine TP; 2'-Deoxy-2'-a-thiomethoxyadenosine TP; 2'-Deoxy-2'-b-aminoadenosine TP; 2'-Deoxy- 2'-b-azidoadenosine TP; 2'-Deoxy-2'-b-bromoadenosine TP; 2'-Deoxy-2'-b-chloroadenosine TP; 2'-Deoxy-2'-b-fluoroadenosine TP; 2'-Deoxy-2'-b-iodoadenosine TP; 2'-Deoxy-2'-b- mercaptoadenosine TP; 2'-Deoxy-2'-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP; 2- lodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine; 2-methylthio-adenine; 2- Trifluoromethyladenosine TP; 3 -Deaza-3 -bromoadenosine TP; 3 -Deaza-3 -chloroadenosine TP; 3 -Deaza-3 -fluoroadenosine TP; 3 -Deaza-3 -iodoadenosine TP; 3 -Deazaadenosine TP; 4'- Azidoadenosine TP; 4'-Carbocyclic adenosine TP; 4'-Ethynyladenosine TP; 5 '-Homoadenosine TP; 8-Aza-ATP; 8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9- Deazaadenosine TP; 2-aminopurine; 7-deaza-2,6-diaminopurine; 7-deaza-8-aza-2,6- diaminopurine; 7-deaza-8-aza-2-aminopurine; 2,6-diaminopurine; 7-deaza-8-aza-adenine, 7- deaza-2-aminopurine; 2-thiocytidine; 3 -methylcytidine; 5 -formylcytidine; 5- hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine; 2'-O-methylcytidine; 2'-O- methylcytidine; 5,2'-O-dimethylcytidine; 5-formyl-2'-O-methylcytidine; Lysidine; N4,2'-O- dimethylcytidine; N4-acetyl-2'-O-methylcytidine; N4-methylcytidine; N4,N4-Dimethyl-2'- OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine; Pseudo-iso-cytidine; pyrrolo-cytidine; a- thio-cytidine; 2-(thio)cytosine; 2'-Amino-2'-deoxy-CTP; 2'-Azido-2'-deoxy-CTP; 2'-Deoxy- 2'-a-aminocytidine TP; 2'-Deoxy-2'-a-azidocytidine TP; 3 (deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine; 3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2'-O- dimethylcytidine; 5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5 (trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine; 5-(halo)cytosine; 5- (propynyl)cytosine; 5-(trifluoromethyl)cytosine; 5-bromo-cytidine; 5-iodo-cytidine; 5- propynyl cytosine; 6-(azo)cytosine; 6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine; 1-methyl-l-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine; 2- methoxy-5-methyl-cytidine; 2-methoxy-cytidine; 2-thio-5-methyl-cytidine; 4-methoxy-l- methyl-pseudoisocytidine; 4-methoxy-pseudoisocytidine; 4-thio-l -methyl- 1-deaza- pseudoisocytidine; 4-thio- 1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine; 5-aza- zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine; Zebularine; (E)-5-(2-Bromo- vinyl)cytidine TP; 2,2'-anhydro-cytidine TP hydrochloride; 2'Fluor-N4-Bz-cytidine TP;2'Fluoro-N4-Acetyl-cytidine TP; 2'-O-Methyl-N4-Acetyl-cytidine TP; T0-methyl-N4-Bz- cytidine TP; 2'-a-Ethynylcytidine TP; 2'-a-Trifluoromethylcytidine TP; 2'-b-Ethynylcytidine TP; 2'-b-Trifluoromethylcytidine TP; 2'-Deoxy-2',2'-difluorocytidine TP; 2'-Deoxy-2'-a- mercaptocytidine TP; 2'-Deoxy-2'-a-thiomethoxycytidine TP; 2'-Deoxy-2'-b-aminocytidine TP; 2'-Deoxy-2'-b-azidocytidine TP; 2'-Deoxy-2'-b-bromocytidine TP; 2'-Deoxy-2'-b- chlorocytidine TP; 2'-Deoxy-2'-b-fluorocytidine TP; 2'-Deoxy-2'-b-iodocytidine TP; 2'- Deoxy-2'-b-mercaptocytidine TP; 2'-Deoxy-2'-b-thiomethoxycytidine TP; 2'-O-Methyl-5-(l- propynyl)cytidine TP; 3'-Ethynylcytidine TP; 4'-Azidocytidine TP; 4'-Carbocyclic cytidine TP; 4'-Ethynylcytidine TP; 5-(l-Propynyl)ara-cytidine TP; 5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidine TP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5- Ethynylara-cytidine TP; 5-Ethynylcytidine TP; 5 '-Homo-cytidine TP; 5-Methoxy cytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidine TP; Pseudoisocytidine; 7-m ethylguanosine; N2,2'-O-dimethylguanosine; N2-methylguanosine; Wyosine; l,2'-O-dimethylguanosine; 1 -methylguanosine; 2'-O-methylguanosine; 2'-O- ribosylguanosine (phosphate); 3 '-O-methylguanosine; 2'-O-ribosylguanosine (phosphate); 7- aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine; Methylwyosine; N2,7-dimethylguanosine; N2,N2,2'-O-trimethylguanosine; N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine; N2,7,2'-O-trimethylguanosine; 6-thio-guanosine; 7-deaza- guanosine; 8-oxo-guanosine; Nl-methyl-guanosine; a-thio-guanosine; 2 (propyl)guanine; 2- (alkyl)guanine; 2'-Amino-2'-deoxy-GTP; 2'-Azido-2'-deoxy-GTP; 2'-Deoxy-2'-a- aminoguanosine TP; 2'-Deoxy-2'-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7 (deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine; 7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8 (halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine; 8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine; 8-(hydroxyl)guanine; 8- (thioalkyl)guanine; 8-(thiol)guanine; aza guanine; deaza guanine; N (methyl)guanine; N- (methyl)guanine; l-methyl-6-thio-guanosine; 6-methoxy -guanosine; 6-thio-7-deaza-8-aza- guanosine; 6-thio-7-deaza-guanosine; 6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine; N2-methyl-6-thio-guanosine; 1-Me-GTP; 2'Fluoro-N2-isobutyl-guanosine TP; 2'O-methyl-N2-isobutyl-guanosine TP; 2'-a- Ethynylguanosine TP; 2'-a-Trifluoromethylguanosine TP; 2'-b-Ethynylguano sine TP; 2'-b- Trifluorom ethylguanosine TP; 2'-Deoxy-2',2'-difluoroguanosine TP; 2'-Deoxy-2'-a-mercaptoguanosine TP; 2'-Deoxy-2'-a-thiomethoxyguanosine TP; 2'-Deoxy-2'-b- aminoguanosine TP; 2'-Deoxy-2'-b-azidoguanosine TP; 2'-Deoxy-2'-b-bromoguanosine TP; 2'-Deoxy-2'-b-chloroguanosine TP; 2'-Deoxy-2'-b-fluoroguanosine TP; 2'-Deoxy-2'-b- iodoguanosine TP; 2'-Deoxy-2'-b-mercaptoguanosine TP; 2'-Deoxy-2'-b- thiomethoxyguanosine TP; 4'-Azidoguanosine TP; 4'-Carbocyclic guanosine TP; 4'- Ethynylguanosine TP; 5 '-Homo-guanosine TP; 8-bromo-guanosine TP; 9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1 -methylinosine; Inosine; l,2'-O-dimethylinosine; 2'-O- methylinosine; 7-methylinosine; 2'-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine; Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deaza thymidine; deoxy-thymidine; 2'-O-methyluridine; 2-thiouridine; 3 -methyluridine; 5- carboxymethyluridine; 5-hydroxyuridine; 5-methyluridine; 5-taurinomethyl-2-thiouridine; 5- taurinom ethyluridine; Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine; l-methyl-3-(3-amino-5-carboxypropyl)pseudouridine; 1-methylpseduouridine; 1-ethyl- pseudouridine; 2'-O-methyluridine; 2'-O-methylpseudouridine; 2'-O-methyluridine; 2-thio-2'- O-methyluridine; 3-(3-amino-3-carboxypropyl)uridine; 3,2'-O-dimethyluridine; 3-Methyl- pseudo-Uridine TP; 4-thiouridine; 5-(carboxyhydroxymethyl)uridine; 5- (carboxyhydroxymethyl)uridine methyl ester; 5,2'-O-dimethyluridine; 5,6-dihydro-uridine; 5- aminomethyl-2-thiouridine; 5-carbamoylmethyl-2'-O-methyluridine; 5- carbamoylmethyluridine; 5-carboxyhydroxymethyluridine; 5-carboxyhydroxymethyluridine methyl ester; 5-carboxymethylaminomethyl-2'-O-methyluridine; 5- carboxymethylaminomethyl-2 -thiouridine; 5-carboxymethylaminomethyl-2-thiouridine; 5- carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine; 5- Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2'-O-methyluridine; 5- methoxycarbonylmethyl-2 -thiouridine; 5-methoxy carbonylmethyluridine; 5-methyluridine,), 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5- methylaminomethyl-2-thiouridine; 5-methylaminomethyluridine; 5-Methyldihydrouridine; 5- Oxyacetic acid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP; Nl-methyl-pseudo- uracil; Nl-ethyl-pseudo-uracil; uridine 5-oxyacetic acid; uridine 5-oxyacetic acid methyl ester; 3-(3-Amino-3-carboxypropyl)-Uridine TP; 5-(iso-Pentenylaminomethyl)-2-thiouridine TP; 5- (iso-Pentenylaminomethyl)-2'-O-methyluridine TP; 5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil; a-thio-uridine; 1 (aminoalkylamino-carbonylethylenyl)-2(thio)- pseudouracil; 1 (aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1 (aminocarbonylethylenyl)-2(thio)- pseudouracil; 1 (aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonylethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted 2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1 substituted pseudouracil; l-(aminoalkylamino-carbonylethylenyl)-2- (thio)-pseudouracil; l-Methyl-3 -(3 -amino-3 -carboxypropyl) pseudouridine TP; l-Methyl-3- (3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2' deoxy uridine; 2' fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2' methyl, 2'amino, 2'azido, 2 'fluoro-guanosine; 2'-Amino-2'-deoxy-UTP; 2'-Azido-2'-deoxy- UTP; 2'-Azido-deoxyuridine TP; 2'-O-methylpseudouridine; 2' deoxy uridine; 2' fluorouridine; 2'-Deoxy-2'-a-aminouridine TP; 2'-Deoxy-2'-a-azidouridine TP; 2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4 (thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4- thiouracil; 5 (l,3-diazole-l-alkyl)uracil; 5 (2-aminopropyl)uracil; 5 (aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5 (guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2- (thio)uracil; 5 (methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl) 2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2 (thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; (methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5 (trifluoromethyl)uracil; 5-(2-aminopropyl)uracil; 5-(alkyl)-2- (thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil; 5-(alkyl)-4 (thio)pseudouracil; 5- (alkyl)pseudouracil; 5-(alkyl)uracil; 5-(alkynyl)uracil; 5-(allylamino)uracil; 5- (cyanoalkyl)uracil; 5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil; 5- (guanidiniumalkyl)uracil; 5-(halo)uracil; 5-(l,3-diazole-l-alkyl)uracil; 5 -(m ethoxy )uracil; 5- (methoxycarbonylmethyl)-2-(thio)uracil; 5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl) 2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil; 5-(methyl)-2- (thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil; 5-(methyl)-4 (thio)pseudouracil; 5- (methyl)pseudouracil; 5-(methylaminomethyl)-2 (thio)uracil; 5-(methylaminomethyl)- 2,4(dithio)uracil; 5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil; 5- (trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine; 5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine; allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-l-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP; 1- carboxymethyl-pseudouridine; 1 -methyl- 1-deaza-pseudouri dine; 1-propynyl-uridine; 1-taurinom ethyl- 1 -methyl-uridine; 1 -taurinom ethyl-4-thio-uri dine; 1 -taurinom ethylpseudouridine; 2-methoxy-4-thio-pseudouridine; 2-thio-l -methyl- 1-deaza-pseudouri dine; 2- thio-l-methyl-pseudouridine; 2-thio-5-aza-uridine; 2-thio-dihydropseudouridine; 2-thio- dihydrouridine; 2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine; 4-methoxy- pseudouridine; 4-thio-l-methyl-pseudouridine; 4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine; (±)l-(2-Hydroxypropyl)pseudouridine TP; (2R)-l-(2- Hydroxypropyl)pseudouridine TP; (2S)-l-(2-Hydroxypropyl)pseudouridine TP; (E)-5-(2- Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP; (Z)-5-(2-Bromo-vinyl)ara- uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP; l-(2,2,2-Trifluoroethyl)-pseudo-UTP; 1- (2,2,3,3,3-Pentafluoropropyl)pseudouridine TP; l-(2,2-Diethoxyethyl)pseudouridine TP; 1- (2,4,6-Trimethylbenzyl)pseudouridine TP; l-(2,4,6-Trimethyl-benzyl)pseudo-UTP; l-(2,4,6- Trimethyl-phenyl)pseudo-UTP; l-(2-Amino-2-carboxyethyl)pseudo-UTP; l-(2-Amino- ethyl)pseudo-UTP; l-(2-Hydroxyethyl)pseudouridine TP; 1 -(2 -Methoxy ethyl)pseudouri dine TP; l-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP; l-(3,4-Dimethoxybenzyl)pseudouridine TP; l-(3-Amino-3-carboxypropyl)pseudo-UTP; l-(3- Amino-propyl)pseudo-UTP; l-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP; l-(4-Amino-4- carboxybutyl)pseudo-UTP; 1 -(4- Amino-benzyl)pseudo-UTP; 1 -(4- Amino-butyl)pseudo-UTP; l-(4-Amino-phenyl)pseudo-UTP; l-(4-Azidobenzyl)pseudouridine TP; l-(4- Bromobenzyl)pseudouridine TP; l-(4-Chlorobenzyl)pseudouridine TP; l-(4- Fluorobenzyl)pseudouridine TP; l-(4-Iodobenzyl)pseudouridine TP; l-(4- Methanesulfonylbenzyl)pseudouridine TP; l-(4-Methoxybenzyl)pseudouridine TP; l-(4- Methoxy-benzyl)pseudo-UTP; l-(4-Methoxy-phenyl)pseudo-UTP; l-(4-Methylbenzyl)pseudouridine TP; l-(4-Methyl-benzyl)pseudo-UTP; l-(4- Nitrobenzyl)pseudouridine TP; l-(4-Nitro-benzyl)pseudo-UTP; l(4-Nitro-phenyl)pseudo- UTP; l-(4-Thiomethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethoxybenzyl)pseudouridine TP; l-(4-Trifluoromethylbenzyl)pseudouridine TP; 1- (5-Amino-pentyl)pseudo-UTP; l-(6-Amino-hexyl)pseudo-UTP; 1,6-Dimethyl-pseudo-UTP; l-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridine TP; l-{3- [2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP; 1-Acetylpseudouridine TP; 1- Alkyl-6-( 1 -propynyl)-pseudo-UTP; 1 -Alkyl-6-(2-propynyl)-pseudo-UTP; 1 - Alkyl-6-allyl- pseudo-UTP; l-Alkyl-6-ethynyl-pseudo-UTP; l-Alkyl-6-homoallyl-pseudo-UTP; l-Alkyl-6- vinyl-pseudo-UTP; 1 -Allylpseudouridine TP; 1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP; 1 -Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP; 1- Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP; 1-Butyl-pseudo-UTP; 1- Cyanomethylpseudouridine TP; 1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo- UTP; 1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP; 1-Cyclohexylmethyl- pseudo-UTP; 1-Cyclohexyl-pseudo-UTP; 1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl- pseudo-UTP; 1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP; 1- Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP; 1-Ethyl-pseudo-UTP; 1- Hexyl-pseudo-UTP; 1 -Homoallylpseudouridine TP; 1 -Hydroxymethylpseudouridine TP; 1- iso-propyl-pseudo-UTP; l-Me-2-thio-pseudo-UTP; l-Me-4-thio-pseudo-UTP; 1-Me-alpha- thio-pseudo-UTP; 1 -Methanesulfonylmethylpseudouridine TP; 1-Methoxymethylpseudouridine TP; l-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP; 1-Methyl- 6-(4-morpholino)-pseudo-UTP; 1 -Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1 -Methyl-6- (substituted phenyl)pseudo-UTP; l-Methyl-6-amino-pseudo-UTP; l-Methyl-6-azido-pseudo- UTP; l-Methyl-6-bromo-pseudo-UTP; l-Methyl-6-butyl-pseudo-UTP; l-Methyl-6-chloro- pseudo-UTP; l-Methyl-6-cyano-pseudo-UTP; l-Methyl-6-dimethylamino-pseudo-UTP; 1- Methyl-6-ethoxy-pseudo-UTP; 1 -Methyl-6-ethylcarboxylate-pseudo-UTP; 1 -Methyl-6-ethyl- pseudo-UTP; l-Methyl-6-fluoro-pseudo-UTP; l-Methyl-6-formyl-pseudo-UTP; l-Methyl-6- hydroxyamino-pseudo-UTP; 1 -Methyl-6-hydroxy-pseudo-UTP; 1 -Methyl-6-iodo-pseudo- UTP; l-Methyl-6-iso-propyl-pseudo-UTP; l-Methyl-6-methoxy-pseudo-UTP; l-Methyl-6- methylamino-pseudo-UTP; 1 -Methyl-6-phenyl-pseudo-UTP; 1 -Methyl-6-propyl-pseudo- UTP; l-Methyl-6-tert-butyl-pseudo-UTP; l-Methyl-6-trifluoromethoxy-pseudo-UTP; 1- Methyl-6-trifluoromethyl-pseudo-UTP; 1 -Morpholinomethylpseudouridine TP; 1-Pentyl- pseudo-UTP; 1 -Phenyl -pseudo-UTP; 1 -Pivaloylpseudouridine TP; 1 -Propargylpseudouridine TP; 1-Propyl-pseudo-UTP; 1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl- pseudo-UTP; 1 -Thiomethoxymethylpseudouridine TP; 1-Thiomorpholinomethylpseudouridine TP; 1-Trifluoroacetylpseudouridine TP; 1- Trifluoromethyl-pseudo-UTP; 1-Vinylpseudouridine TP; 2,2'-anhydro-uridine TP; 2'-bromo- deoxyuridine TP; 2'-F-5-Methyl-T-deoxy-UTP; 2'-OMe-5-Me-UTP; 2'-OMe-pseudo-UTP; 2'- a-Ethynyluridine TP; 2'-a-Trifluoromethyluridine TP; 2'-b-Ethynyluridine TP; 2'-b- Trifluoromethyluridine TP; 2'-Deoxy-2',2'-difluorouridine TP; 2'-Deoxy-2'-a-mercaptouridine TP; 2'-Deoxy-2'-a-thiomethoxyuridine TP; 2'-Deoxy-2'-b-aminouridine TP; 2'-Deoxy-2'-b- azidouridine TP; 2'-Deoxy-2'-b-bromouridine TP; 2'-Deoxy-2'-b-chlorouridine TP; 2'-Deoxy-2'-b-fluorouridine TP; 2'-Deoxy-2'-b-iodouridine TP; 2'-Deoxy-2'-b-mercaptouridine TP; 2'- Deoxy-2'-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine; 2-methoxyuridine; 2'-0- Methyl-5-(l-propynyl)uridine TP; 3-Alkyl-pseudo-UTP; 4'-Azidouridine TP; 4'-Carbocyclic uridine TP; 4'-Ethynyluridine TP; 5-(l-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridine TP; 5- Cyanouridine TP; 5-Dimethylaminouridine TP; 5 '-Homo-uridine TP; 5-iodo-2'-fluoro- deoxyuridine TP; 5-Phenylethynyluridine TP; 5-Trideuteromethyl-6-deuterouridine TP; 5- Trifluoromethyl-Uridine TP; 5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP; 6- (4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP; 6-(Substituted-Phenyl)- pseudo-UTP; 6-Amino-pseudo-UTP; 6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl- pseudo-UTP; 6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP; 6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP; 6-Fluoro- pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP; 6-Hydroxy-pseudo- UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP; 6-Methoxy-pseudo-UTP; 6- Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo- UTP; 6-Propyl-pseudo-UTP; 6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP; 6- Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine l-(4- methylbenzenesulfonic acid) TP; Pseudouridine l-(4-methylbenzoic acid) TP; Pseudouridine TP l-[3-(2-ethoxy)]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-(2-ethoxy)-ethoxy]- ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy}- ethoxy]-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP l-[3-{2-(2-[2-ethoxy]-ethoxy)- ethoxy}] propionic acid; Pseudouridine TP l-[3-{2-(2-ethoxy)-ethoxy}]propionic acid; Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP 1-methylphosphonic acid diethyl ester; Pseudo-UTP-Nl -3 -propionic acid; Pseudo-UTP-Nl-4-butanoic acid; Pseudo- UTP-Nl-5-pentanoic acid; Pseudo-UTP-Nl -6-hexanoic acid; Pseudo-UTP-Nl -7-heptanoic acid; Pseudo-UTP-Nl -methyl-p-benzoic acid; Pseudo-UTP-Nl -p-benzoic acid; Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine; undermodified hydroxywybutosine; 4- demethylwyosine; 2,6-(diamino)purine; l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl: l,3-(diaza)- 2-(oxo)-phenthiazin-l-yl;l,3-(diaza)-2-(oxo)-phenoxazin-l-yl;l,3,5-(triaza)-2,6-(dioxa)- naphthalene;2 (amino)purine;2,4,5-(trimethyl)phenyl;2' methyl, 2'amino, 2'azido, 2'fluro- cytidine;2' methyl, 2'amino, 2'azido, 2'fluro-adenine;2'methyl, 2'amino, 2'azido, 2'fluro- uridine;2'-amino-T-deoxyribose; 2-amino-6-Chloro-purine; 2-aza-inosinyl; 2'-azido-T- deoxyribose; 2'fluoro-T-deoxyribose; 2'-fluoro-modified bases; 2'-O-methyl-ribose; 2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl; 2-pyridinone; 3 nitropyrrole; 3- (methyl)-7-(propynyl)isocarbostyrilyl; 3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole; 4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl; 5-nitroindole; 6- (aza)pyrimidine; 6-(azo)thymine; 6-(methyl)-7-(aza)indolyl; 6-chloro-purine; 6-phenyl- pyrrolo-pyrimidin-2-on-3-yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l- yl; 7-(aminoalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(aminoalkylhydroxy)- 1 , 3 -(diaza)-2-(oxo)-phenoxazin- 1 -yl ; 7-(aminoalkylhy droxy)- 1 , 3 -(diaza)-2-(oxo)- phenthiazin- 1 -yl; 7-(aminoalkylhy droxy)- 1 ,3 -(diaza)-2-(oxo)-phenoxazin- 1 -yl; 7-(aza)indolyl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl; 7- (guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 7-(guanidiniumalkyl- hydroxy)-l,3-(diaza)-2-(oxo)-phenthiazin-l-yl; 7-(guanidiniumalkylhydroxy)-l,3-(diaza)-2- (oxo)-phenoxazin-l-yl; 7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl, propynyl- 7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted l-(aza)-2-(thio)-3-(aza)-phenoxazin-l-yl; 7- substituted l,3-(diaza)-2-(oxo)-phenoxazin-l-yl; 9-(methyl)-imidizopyridinyl; Aminoindolyl; Anthracenyl; bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; bis- ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl; Isocarbostyrilyl; Isoguanisine; N2-substituted purines; N6-methyl- 2-amino-purine; N6-substituted purines; N-alkylated derivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl; Nitroindazolyl; Nitropyrazolyl; Nubularine; 06- substituted purines; O-alkylated derivative; ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo- pyrimidin-2-on-3-yl; ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP; para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; para-sub stituted-6- phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl; Phenanthracenyl; Phenyl; propynyl-7- (aza)indolyl; Pyrenyl; pyridopyrimidin-3-yl; pyridopyrimidin-3-yl, 2-oxo-7-amino- pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl; Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted 1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5'-TP; 2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine; pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP; Formycin B TP; Pyrrolosine TP; 2'- OH-ara-adenosine TP; 2'-OH-ara-cytidine TP; 2'-OH-ara-uridine TP; 2'-OH-ara-guanosineTP; 5-(2-carbomethoxyvinyl)uridine TP; and N6-(19-Amino-pentaoxanonadecyl)adenosine TP.

[0155] In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) include a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases.

[0156] In an example embodiment, modified nucleobases in oligonucleotides (e.g., RNA, such as mRNA) are selected from pseudouridine (y), 2-thiouridine (s2U), 4'-thiouridine, 5- methylcytosine, 2-thio-l -methyl- 1-deaza-pseudouri dine, 2-thio-l-methyl-pseudouridine, 2- thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5- methoxyuridine, 2'-O-methyl uridine, 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), a-thio-guanosine, a-thio- adenosine, 5-cyano uridine, 4'-thio uridine 7-deaza-adenine, 1-methyl-adenosine (mlA), 2- methyl-adenine (m2 A), N6-methyl-adenosine (m6A), and 2,6-Diaminopurine, (I), 1 -methylinosine (mil), wyosine (imG), methyl wyosine (mimG), 7-deaza-guanosine, 7-cyano-7-deaza- guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G),1-methyl-guanosine (mlG), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8- dimethyladenosine, 2-geranylthiouridine, 2-lysidine, 2-sel enouridine, 3-(3-amino-3- carboxypropyl)-5,6-dihydrouridine, 3-(3-amino-3-carboxypropyl)pseudouridine, 3- methylpseudouridine, 5-(carboxyhydroxymethyl)-2'-O-methyluridine methyl ester, 5- aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine, 5-aminomethyluridine, 5- carbamoylhydroxymethyluridine, 5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2- thiouridine, 5-carboxymethylaminomethyl-2-geranylthiouridine, 5- carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine, 5-hydroxycytidine, 5- methylaminomethyl-2-geranylthiouridine, 7-aminocarboxypropyl-demethylwyosine, 7- aminocarboxypropylwyosine, 7-aminocarboxypropylwyosine methyl ester, 8- methyladenosine, N4,N4-dimethylcytidine, N6-formyladenosine, N6- hydroxymethyladenosine, agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl- queuosine, methylated undermodified hydroxywybutosine, N4,N4,2'-O-trimethylcytidine, geranylated 5-methylaminomethyl-2-thiouridine, geranylated 5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQObase, preQlbase, and combinations of two or more thereof. In anexample embodiment, at least one chemically modified nucleoside is selected from pseudouridine, 1-methyl-pseudouridine, 1-ethyl-pseudouridine, 5-methylcytosine, 5- methoxyuridine, and a combination thereof. In an example embodiment, the polyribonucleotide (e.g., RNA polyribonucleotide, such as mRNA polyribonucleotide) includes a combination of at least two (e.g., 2, 3, 4 or more) of the aforementioned modified nucleobases. In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) include a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases.

[0157] In an example embodiment, modified nucleobases in oligonucleotides (e.g., RNA, such as mRNA) are selected from 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (ely), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine (y), a-thio- guanosine and a-thio-adenosine. In an example embodiment, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4, or more) of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0158] In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) comprise pseudouridine (y) and 5-methyl-cytidine (m5C). In an example embodiment, the oligoribonucleotides (e.g., RNA, such as mRNA) comprise 1-methyl-pseudouridine (mly). In an example embodiment, the oligoribonucleotides (e.g., RNA, such as mRNA) comprise 1- ethyl-pseudouridine (ely). In an example embodiment, the oligoribonucleotides (e.g., RNA, such as mRNA) comprise 1-methyl-pseudouridine (mly) and 5-methyl-cytidine (m5C). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 1-ethyl- pseudouridine (ely) and 5-methyl-cytidine (m5C). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 2-thiouridine (s2U). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 2-thiouridine and 5-methyl-cytidine (m5C). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise methoxy-uridine (mo5U). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 5-methoxy-uridine (mo5U) and 5- methyl-cytidine (m5C). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 2'-O-methyl uridine. In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise 2'-O-methyl uridine and 5-methyl-cytidine (m5C). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise N6-methyl-adenosine (m6A). In an example embodiment, the polyribonucleotides (e.g., RNA, such as mRNA) comprise N6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

[0159] In an example embodiment, oligonucleotides (e.g., RNA, such as mRNA) are uniformly modified (e.g., fully modified throughout the entire sequence) for a particular modification. For example, a polynucleotide can be uniformly modified with 1-methyl- pseudouridine, meaning all uridine residues in the mRNA sequence are replaced with 1-methyl- pseudouridine. Similarly, a polynucleotide can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0160] Exemplary nucleobases and nucleosides having a modified cytosine include N4- acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and 2- thio-5-methyl-cytidine.

[0161] In an example embodiment, a modified nucleobase is a modified uridine. Exemplary nucleobases and nucleosides having a modified uridine include 1-methyl- pseudouridine (mlv), 1-ethyl-pseudouridine (ely), 5-methoxy uridine, 2-thio uridine, 5-cyano uridine, 2'-O-methyl uridine and 4'-thio uridine.

[0162] In an example embodiment, a modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 7-deaza-adenine, 1 -methyl-adenosine (ml A), 2-methyl-adenine (m2A), and N6-methyl-adenosine (m6A).

[0163] In an example embodiment, a modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1- methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 7-deaza-guanosine, 7-cyano- 7-deaza-guanosine (preQO), 7-aminomethyl-7-deaza-guanosine (preQi), 7-methyl-guanosine (m7G), 1-methyl-guanosine (mlG), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.

[0164] The oligonucleotides of the present disclosure may be partially or fully modified along the entire molecule length. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a polynucleotide of the invention, or each predetermined sequence region thereof (e.g., in the mRNA including or excluding the polyA tail). In an example embodiment, all nucleotides X in a polynucleotide of the present disclosure (or in a given sequence region thereof) aremodified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0165] The oligonucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that the presence of unmodified A, G, U, or C accounts for any remaining percentage.

[0166] The oligonucleotides may contain at a minimum of 1% and a maximum of 100% modified nucleotides or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the oligonucleotides may contain a modified pyrimidine, such as a modified uracil or cytosine. In an example embodiment, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the uracil in the oligonucleotide is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound with a single unique structure or by a plurality of compounds with different structures (e.g., 2, 3, 4, or more unique structures). In an example embodiment, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90%, or 100% of the cytosine in the oligonucleotide is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound with a single unique structure or by a plurality of compounds with different structures (e.g., 2, 3, 4, or more unique structures).

[0167] In an example embodiment, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine(s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5 -iodo-uridine or 5-bromo-uridine), 3- methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5- methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (rm5U), 1 -taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1 -taurinomethyl-4- thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1- methyl-pseudouridine (m1!] / ), 1-ethyl-pseudouridine (ely), 5-methyl-2 -thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m y), 4-thio-l-methyl-pseudouridine, 3-methyl- pseudouridine (m3y), 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2- thio-1 -methyl- 1-deaza-pseudouri dine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3y), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio- uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (vm), 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl- uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O- dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1- thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino)]uridine.

[0168] In an example embodiment, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6- aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (FC), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocyti dine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, lysidine (I C), a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O- dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl- cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (FCm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1 -thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0169] In an example embodiment, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8- aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6- diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenosine (nfA), 2-methyl- adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis- hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl- adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl- adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6- acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio- adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m6Am), N6,N6,2'-O- trimethyl-adenosine (m62Am), l,2'-O-dimethyl-adenosine (m'Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1 -thio-adenosine, 8-azido-adenosine, 2'-F- ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)- adenosine.

[0170] In an example embodiment, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m1!), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxy wybutosine (02yW), hydroxy wybutosine (OhyW), undermodified hydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7- deaza-guanosine (preQo), 7-aminomethyl-7-deaza-guanosine (preQi), archaeosine (G+), 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6- methoxy-guanosine, 1-methyl-guanosine (nfG), N2-methyl-guanosine (m2G), N2,N2- dimethyl-guanosine (m22G), N2,7-dimethyl-guano sine (m2-7G), N2,N2,7-dimethyl-guanosine (m2’2’7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2- methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio-guanosine, 2'-O-methyl- guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m2Gm), N2,N2-dimethyl-2'-O-methyl- guanosine (m22Gm), l-methyl-2'-O-methyl-guanosine (m'Gm), N2,7-dimethyl-2'-O-methyl- guanosine (m2’7Gm), 2'-O-methyl-inosine (Im), l,2'-O-dimethyl-inosine (nflm), 2'-O- ribosylguanosine (phosphate) (Gr(p)), 1 -thio-guanosine, 06-methyl-guanosine, 2'-F-ara- guanosine, and 2'-F-guanosine.Oligonucleotide and LP Complexation and Formation

[0171] In an example embodiment, at least one oligonucleotide is complexed or associated with a lipid particle as described herein, including lipid particles comprising the compounds of Formula I. The complexation process involves the association of negatively charged oligonucleotide molecules with positively charged or ionizable cationic lipids to form lipid particles through self-assembly.

[0172] In an example embodiment, the oligonucleotide is an mRNA molecule complexed or associated with a lipid particle as described herein. In an example embodiment, the mRNA is complexed or associated with lipids to form lipid particles comprising a cationic lipid, a neutral lipid, a sterol, and a polymer-conjugated lipid as described in the lipid particle sections above. In an example embodiment, at least one mRNA is complexed with the compounds of Formula I, wherein the cationic lipid component is the compounds of Formula I. In an example embodiment, the mRNA is complexed with a lipid particle comprising Compound 1 as the cationic lipid component.Mechanism of Complex Formation

[0173] In an example embodiment, mRNA-lipid complexes form via electrostatic interactions between the mRNA's negatively charged phosphate backbone and the cationic lipid's positively charged or protonated amine groups. At acidic pH, ionizable cationic lipids such as the compound of Formula I become protonated and carry a positive charge, facilitating electrostatic attraction to the anionic mRNA. This charge-charge interaction drives the initial association between mRNA and lipid components.

[0174] In an example embodiment, the complexation process involves the self-assembly of lipid components around the mRNA cargo during the mixing of an aqueous mRNA solution with an organic lipid solution. When the two solutions are combined under controlled conditions, the lipids spontaneously organize into structured particles that encapsulate the mRNA. Both electrostatic interactions between charged species and hydrophobic interactions among lipid components drive self-assembly.

[0175] In an example embodiment, multiple lipid molecules associate with a single mRNA molecule to form a complex. The stoichiometry of lipid-to-mRNA association depends on several factors, including the N / P ratio (the molar ratio of nitrogen atoms in the cationic lipid to phosphate groups in the mRNA), the lipid composition, the pH of the formulation buffer, and the ionic strength of the medium.Encapsulation and Particle Morphology

[0176] In an example embodiment, the oligonucleotide is encapsulated within the interior aqueous core of the lipid particle, which is surrounded by a lipid bilayer or multilayer. In an example embodiment, the encapsulation process occurs during the formation step when the aqueous and organic phases are rapidly mixed, trapping the oligonucleotide within the forming lipid structures. The rapid mixing ensures homogeneous particle formation and efficient mRNA encapsulation.

[0177] The term "encapsulation" as used herein refers to the incorporation of oligonucleotides, including mRNA, into lipid particles such that the nucleic acid is substantially contained within the lipid particle's interior, protected from the external environment. In an example embodiment, the LP-incorporated nucleic acids may be entirely or partially located within the lipid particle's interior space, within the lipid particle bilayer membrane, or associated with the lipid particle's exterior surface. In an example embodiment,the mRNA is substantially encapsulated within the interior aqueous compartment of the lipid particle.

[0178] According to an example embodiment, the mRNA is complexed or associated with lipids to form one or more liposomes, lipoplexes, lipid particles, or lipid nanoparticles. The term "lipid particle" as used herein is not restricted to any particular morphology. It includes any morphology generated when a cationic lipid and, optionally, one or more additional lipids are combined in an aqueous environment in the presence of an RNA. Possible morphologies include, without limitation, liposomes, lipid complexes, lipoplexes, emulsions, micelles, lipidic capsules, and suspensions.Formation Methods and Process Conditions

[0179] In an example embodiment, oligonucleotide-lipid complexes are formed by mixing an aqueous solution containing the oligonucleotide with an organic solution containing lipid components. The aqueous solution typically comprises mRNA dissolved in an aqueous buffer, while the organic solution comprises lipid components dissolved in an alcohol or another water-miscible organic solvent.

[0180] In an example embodiment, the aqueous buffer is selected from citrate buffer, phosphate buffer, acetate buffer, or succinate buffer. In an example embodiment, the buffer has a pH of about 3.0 to about 7.0, or about 3.5 to about 6.5, or about 4.0 to about 6.0, or about 4.0 to about 5.5. The acidic pH facilitates protonation of ionizable cationic lipids, promoting their interaction with the anionic mRNA. In an example embodiment, the aqueous buffer comprises 1 mM citrate and 150 mM NaCl at pH 4.5.

[0181] In an example embodiment, the buffer may contain salts such as sodium chloride, potassium chloride, calcium salts, or combinations thereof. The ionic strength of the buffer can influence electrostatic interactions between mRNA and lipids, thereby affecting particle formation and stability.

[0182] In an example embodiment, the organic solution comprises an alcohol. In an example embodiment, the alcohol is ethanol, methanol, isopropanol, or a combination thereof. In an example embodiment, the alcohol is ethanol. The organic solvent dissolves the lipid components, facilitating their mixing with the aqueous mRNA solution.

[0183] In an example embodiment, the mixing of aqueous and organic phases is performed using controlled mixing methods that ensure rapid and homogeneous mixing. Rapid mixing is important for forming uniform particles with a consistent size distribution and highencapsulation efficiency. In an example embodiment, the mixing is performed using microfluidic mixing devices, T-junction mixers, staggered herringbone mixers, or other devices that provide controlled laminar or turbulent mixing.

[0184] In an example embodiment, the aqueous and organic solutions are mixed at defined flow rates and flow rate ratios. The flow rate ratio (aqueous: organic) can affect particle size, encapsulation efficiency, and particle homogeneity. In an example embodiment, the flow rate ratio is about 1 : 1, about 2: 1, about 3: 1, about 4: 1, or about 5:1 (aqueous: organic). In an example embodiment, the flow rate ratio is about 3:1.

[0185] In an example embodiment, mixing occurs under controlled temperature conditions. The temperature during mixing can influence lipid fluidity, self-assembly kinetics, and final particle characteristics. In an example embodiment, mixing is performed at room temperature (about 20-25°C). In an example embodiment, mixing is performed at reduced temperature (about 2-10°C) or elevated temperature (about 30-40°C).Post-Formation Processing

[0186] In an example embodiment, following the initial mixing and particle formation, the oligonucleotide-lipid complexes undergo buffer exchange to remove the organic solvent and adjust the external buffer composition. Buffer exchange may be performed using dialysis, tangential flow filtration (TFF), diafiltration, or ultrafiltration. In an example embodiment, the particles are exchanged into a physiologically relevant buffer such as phosphate-buffered saline (PBS), saline, or other pharmaceutically acceptable buffers.

[0187] In an example embodiment, the oligonucleotide-lipid complexes are concentrated following buffer exchange to achieve a desired oligonucleotide concentration for storage and administration. Concentration may be performed by ultrafiltration, centrifugation, or other concentration methods.

[0188] In an example embodiment, the formed and processed mRNA-lipid particles are stored under conditions that maintain particle stability and mRNA integrity. In an example embodiment, the particles are stored at -80°C, -20°C, 2-8°C, or room temperature, depending on the formulation and intended storage duration. In an example embodiment, the particles are stored frozen at -80°C.Factors Affecting Complex Formation

[0189] In an example embodiment, the characteristics of the formed oligonucleotide-lipid complexes depend on multiple formulation parameters. These parameters include, withoutlimitation: the N / P ratio (ratio of cationic lipid nitrogen to mRNA phosphate), the lipid composition and molar ratios, the pH of the aqueous buffer, the ionic strength of the buffer, the concentration of oligonucleotide, the concentration of lipids, the mixing method and flow rates, the temperature during formation, and the organic solvent type and concentration.

[0190] In an example embodiment, these parameters are optimized to achieve desired particle characteristics, including particle size, polydispersity, encapsulation efficiency, surface charge, and colloidal stability. Optimal parameters may vary depending on the specific mRNA cargo, lipid composition, intended application, and target tissue or cell type.

[0191] In an example embodiment, the N / P ratio is selected to achieve complete complexation of the mRNA with lipids while minimizing excess free cationic lipid, which could contribute to toxicity. In an example embodiment, the lipid composition is selected to provide both effective encapsulation during formation and efficient intracellular delivery after administration. In an example embodiment, the mixing conditions are optimized to produce particles with a narrow size distribution and high batch-to-batch reproducibility.

[0192] According to an example embodiment, the oligonucleotide payment, optionally formulated as a pharmaceutical composition or vaccine, is complexed or associated with lipids (in particular, cationic and / or neutral lipids) to form one or more liposomes, lipoplexes, lipid particles, or nanoliposomes, including compounds of Formula I, disclosed herein.

[0193] In an example embodiment, lipid particles (LPs) comprise (a) at least one mRNA, , (b) a cationic lipid, (c) polymer conjugated-lipids (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and (e) optionally, a sterol.

[0194] In an example embodiment, LPs comprise, in addition to the at least one mRNA, optionally comprised by the pharmaceutical composition or vaccine as defined herein, (i) at least one compound of Formula I; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, in a molar ratio of about 20-60 mol% cationic lipid; about 5-25 mol% neutral lipid; about 25-55 mol% sterol; and about 0.5-15% PEG-lipid.EXAMPLE VACCINE COMPOSITIONSCrimean-Congo Hemorrhagic Fever (CCHFV) Vaccine

[0195] In one aspect, an embodiment is directed to a vaccine composition for the treatment of Crimean-Congo Hemorrhagic Fever (CCHFV). The CCHFV RNA vaccine comprises anmRNA construct as described herein, complexed with lipid nanoparticles using the compounds disclosed herein.

[0196] In one embodiment, the mRNA comprises in a 5’ to 3’ direction a 5’ cap modification, a 5’ UTR, the ORF encoding the antigen, a 3’- UTR and a poly-A tail. The 5’ cap modification may comprise any of the modifications described above. In one embodiment, the 5’ cap is a 3’-O-methylguanosine cap. The 5’ and 3’ UTRs may be any UTR described above. In one embodiment, the 5’ UTR is a CCHFV UTR. In one embodiment, the 3’ UTR is a CCHFV UTR. In one embodiment, the 5’ and 3’ UTRs are 5’ and 3’ CCHFV UTRs, respectively.

[0197] In exemplary embodiment, the antigenic polypeptide is a wild-type mucin-modified gp38 (also referred to as GP85). In another embodiment, the antigenic polypeptide is a mucin- modified gp38 mutant (GP85Aglyc), wherein the gp38 sequence has been modified to remove N-glycosylation sites (FIG. 14, FIG. 18, FIG. 19A and B).

[0198] In an embodiment, the mRNA encodes a modified CCHFV nucleocapsid protein comprising an AEVA motif in place of the wild-type DEVD motif at amino acid positions 266- 269. In an embodiment. In an embodiment, the vaccine composition comprises a mRNA construct encoding the mucin-modified gp38 mutant (GP85Aglyc) and a mRNA construct encoding the AEVA mutated nucleocapsid.

[0199] The lipid nanoparticle may be any of the lipid nanoparticles described above. In one embodiment, the lipid nanoparticle comprises heptadecan-9-yl 7-((7-(heptyloxy)-7- oxoheptyl)(2-hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof. The compound is referred to herein as Compound 1 or BP- 104.

[0200] In one embodiment, the CCHFV RNA vaccine is a lipid particle composition comprising one or more neutral lipids, steroids, or polymer-conjugated lipids. In one embodiment, one or more neutral lipids are selected from DSPC, DPPC, DMPC, DOPC,POPC, DOPE, and SM. In an embodiment, wherein the neutral lipid is DSPC. In an embodiment, a molar ratio of compound to neutral lipid is about 2: 1 to about 8:1. In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of compound to steroid is about 5: 1 to about 1 : 1. In one embodiment, the one or more polymer-conjugated lipids comprise one or more pegylated lipids. In one embodiment, the molar ratio of compound to pegylated lipid is about 100: 1 to about 20: 1.

[0201] In one embodiment, the one or more pegylated lipids are selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, PEG dialkyoxypropylcarbamate, or a combination thereof.

[0202] In one embodiment, the one or more pegylated lipids is a compound of Formula (II)

[0203] wherein R1and R2are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds and n has a mean value of about 30 to about 60. In an embodiment, R1and R2are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In an embodiment, n has a mean value of about 45.

[0204] In one embodiment, the one or more pegylated lipids further comprises 1- monomethoxypolyethyleneglycol-2,3-dimyristylglycerol.Cancer Vaccine

[0205] The compositions of the present invention can be used in preparation of cancer vaccines. Cancer RNA vaccines can be administered prophylactically or therapeutically to healthy individuals as part of an active immunization program or during active cancer, either early in the tumor or after the onset of symptoms. In an example embodiment, the amount of an RNA vaccine of the present disclosure provided to a cell, tissue, or subject can be an amount effective for immune control.

[0206] The cancer RNA vaccine can be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound can be an adjuvant or booster. The application time between the initial application of the prophylactic composition and the booster can be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 1 hour, day, 6 days, weeks, 1 week, 10 days, 2 weeks, 3 months, 4 months, 5 months, 6 months, 7 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years, or more than 99 years. In exemplary embodiments, the application time between the initial application of the prophylactic composition and the booster can be but is not limited to one week, two weeks, three weeks, one month, two months, three months, six months, or one year. In an example embodiment, the composition may be administered intramuscularly or intradermally, as with vaccines known in the art.

[0207] mRNA cancer vaccines can be used in various settings depending on the severity of the cancer or the degree of unmet medical needs. As a non-limiting example, mRNA cancer vaccines can treat cancer at any stage. mRNA cancer vaccines have superior properties because they elicit much higher antibody titers and T-cell responses, and these responses occur much earlier than those of commercially available anti-cancer vaccines. Without wishing to be bound by theory, mRNA cancer vaccines are better designed to produce the appropriate protein conformation upon translation, as mRNA cancer vaccines enlist native cellular machinery, unlike traditional vaccines, which are made ex vivo and can trigger undesirable cellular responses. mRNA cancer vaccines are presented to the cellular system more naturally.

[0208] Cancer cells may differentially express cell-surface molecules during distinct phases of tumor progression. For example, cancer cells may express a cell surface antigen benignly but downregulate it after metastasis. Thus, it is contemplated that a tumor antigen or cancer antigen may encompass antigens produced during any stage of cancer progression. The process of the present invention can be adjusted to accommodate these variations. For example, several mRNA vaccines can be generated for a particular patient. For example, the first vaccine may be used at the beginning of the treatment. At a later point in time, a new mRNA vaccine can be generated and administered to the patient to account for the different antigens expressed.

[0209] In an example embodiment, the tumor antigen is one of the following antigens: CD, CD137, 4-IBB, 5T, AGS-5, AGS-16, Angiopoietin 2 (Angiopoietin 2), B7.1, B7.2, B7H, BT- 062, BTLA, CAIX, carcinoembryonic antigen, CTLA, Cripto, ED-, ErbB, EGFL, EpCAM, EphA, EphB, FAP, Fibronectin (Fibronectin), folate receptor, Ganglioside GM (Ganglioside GM), GD, glucocorticoid-induced tumor necrosis factor receptor (GITR), gOOpl, gpA, GPNMB, ICNMB, IGF1, integrin av (integrin), integrin a v P, MULAG-3, MUY-3, MUTIN, MUT-4, MUC, MUX, MUC-4, NOX, NOT-C, NOT-4, NOT, CTD, NOT-4, VIP, CTD, VIA, VIP, VI, PDL1, PSCA, PSMA, RANKL, ROR1, ROR2, SLC44A4, cohesin-1 (Syndecano-1), TACI, TAG-72, Tenascin (Tenascin), TIM3, TRAILR1, TRAILR2, VEGFR-1, VEGFR-2, VEGFR-3, and variants thereof.

[0210] Cancer or tumor includes, but is not limited to, a neoplasm, a malignancy, metastatic cancer, or any disease or condition characterized by uncontrolled growth of cells, such that it will be considered to be cancerous. The cancer may be primary or metastatic. Specific cancers that may be treated according to the invention include, but are not limited to, those listed below (for a review of the conditions, see Fisherman et al., 1985, Medicine, 2nd edition, J.B... Lippincott Co., Philadelphia). Cancers include but are not limited to, biliary tract cancer; bladder cancer; brain cancer, including glioblastoma and medulloblastoma; breast cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial cancer; esophageal cancer; gastric cancer; hematological neoplasms, including acute lymphocytic and myelogenous leukemias, multiple myeloma, AIDS-related leukemia, and adult T-cell leukemia-lymphoma; intraepithelial neoplasms, including Bowen’s disease and Paget’s disease; liver cancer, including hepatocellular carcinoma; lung cancer; lymphomas, including Hodgkin’s disease and lymphocytic lymphoma; neuroblastoma; oral cancer, including squamous cell carcinoma; ovarian cancer, including those caused by epithelial, stromal, and germ cells; pancreatic cancer; prostate cancer; rectal cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma, liposarcoma, fibrosarcoma, and osteosarcoma; skin cancers, including melanoma, Kaposi’s sarcoma, basal cell carcinoma, and squamous cell carcinoma; testicular cancer, including germinal tumors such as seminoma, non-seminoma, teratoma, and choriocarcinoma; thyroid cancer, including thyroid adenocarcinoma and medullary carcinoma; renal cancers, including adenocarcinoma and Wilms’ tumor; colorectal cancer; gastroesophageal cancer; head and neck cancer; solid tumors; and urothelial cancer.Commonly encountered cancers include breast, prostate, lung, ovarian, colorectal, and brain cancers.Methods of Use of the mRNA-LP Vaccines

[0211] In another aspect, the present invention provides methods of immunizing a subject against one or more viruses, parasitic infections, or fungal infections. The present invention further provides methods of eliciting an immune response against one or more viruses, parasitic infections, or a fungal infection in a subject. In an example embodiment, the present methods comprise administering to the subject an effective amount of a composition described herein to a subject.

[0212] In an example embodiment, the methods of immunizing provided herein elicit a broadly protective immune response against multiple epitopes within one or more of adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial vims, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever.

[0213] In an example embodiment, the methods of immunizing provided herein elicit a broadly neutralizing immune response against one or more of adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, and yellow fever. In an example embodiment, the immune response comprises an antibody response. Accordingly, in an example embodiment, the composition described herein can offer broad cross-protection against different viruses.

[0214] In one embodiment, the method of immunizing provided herein elicit a neutralizing response against Crimean-Congo hemorrhagic fever.

[0215] In an example embodiment, the composition described herein can offer broad crossprotection against a viral pathogen is selected from Adenovirus, Crimean-Congo hemorrhagic fever virus, Hepacivirus, Hepatitis B virus, Hepatitis delta virus, Hepatovirus A, Human alphaherpesvirus 3, Human papillomavirus, H1N1, H3N2, Influenza A virus, Influenza B virus, Influenza C virus, Influenza D virus, Japanese encephalitis virus, Measles morbillivirus, Mumps orthorubulavirus, Orthohepevirus A, Poliovirus, Rabies lyssavirus, Rotavirus A, Rotavirus B, Rotavirus C, Rotavirus D, Rotavirus F, Rotavirus G, Rotavirus H, Rotavirus I,Rotavirus J, Rubivirus rubellae, Severe acute respiratory syndrome-related coronavirus, Variola virus, and Yellow fever virus.

[0216] In one embodiment, the compositions described herein offer protection against Crimean-Congo hemorrhagic fever virus.

[0217] In an example embodiment, the composition offers cross-protection against a viral pathogen selected from Adenovirus, Crimean-Congo hemorrhagic fever virus, Hepacvirus, Hepatitis B virus, Hepatitis delta virus, Hepatovirus A, Human alphaherpesvirus 3, Human papillomavirus, H1N1, H3N2, Influenza A virus, Influenza B virus, Influenza C virus, Influenza D virus, Japanese encephalitis virus, Measles morbillivirus, Mumps orthorubulavirus, Orthohepevirus A, Poliovirus, Rabies lyssavirus, Rotavirus A, Rotavirus B, Rotavirus C, Rotavirus D, Rotavirus F, Rotavirus G, Rotavirus H, Rotavirus I, Rotavirus J, Rubivirus rubellae, Severe acute respiratory syndrome-related coronavirus, Variola virus, and Yellow fever virus.

[0218] In an example embodiment, the composition offers cross-protection against avian, swine, seasonal, and / or pandemic influenza viruses. The composition provides cross-protection against one or more influenza A, B, or C subtypes in an example embodiment. In an example embodiment, the composition offers cross-protection against multiple strains of influenza A Hl-subtype viruses (e.g., H1N1), influenza A H3-subtype viruses (e.g., H3N2), influenza A H5-subtype viruses (e.g., H5N1), and / or influenza B viruses (e.g., Yamagata lineage, Victoria lineage).

[0219] In an example embodiment, the present invention provides methods of preventing or treating influenza infections by administering the composition of the invention to a subject.

[0220] In an example embodiment, the subject is suffering from or susceptible to a viral infection (e.g., adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever). In one embodiment, the subject is suffering from or susceptible to Crimean- Congo hemorrhagic fever.

[0221] In an example embodiment, a subject is considered to be suffering from infection if the subject is displaying one or more symptoms commonly associated with a viral infection (e.g., adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies,respirator}- syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever). In one embodiment, the subject is considered to be suffering from infection if the subject is displaying one or more symptoms commonly associated with Crimean-Congo hemorrhagic fever.

[0222] In an example embodiment, the subject is known or believed to have been exposed to the virus (e.g. adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory’ syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever). In one embodiment, the subject is known or believed to have been exposed to Crimean-Congo hemorrhagic fever.

[0223] In an example embodiment, a subject is considered to be susceptible to infection if the subject is known or believed to have been exposed to the virus (e.g., adenovirus, Crimean- Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever). In one embodiment, the subject is considered to be susceptible to infection if the subject is known or believed to have been exposed to Crimean-Congo hemorrhagic fever.

[0224] In an example embodiment, a subject is known or believed to have been exposed to the virus if the subject has been in contact with other individuals known or suspected to have been infected with the virus (e.g., adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever) and / or if the subject is or has been present in a location in which infection (e.g., adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, or yellow fever) is known or thought to be prevalent. In one embodiment, the subject is known or believed to have been exposed to the virus if the subject has been in contact with other individuals known or suspected to have been infected with Crimean-Congo hemorrhagic fever. In an embodiment, if the subject is or has been present in a location in which Crimean-Congo hemorrhagic fever is known or thought to be prevalent.

[0225] In an example embodiment, the composition described herein may be administered before or after developing one or more symptoms of the infection. In an example embodiment, the composition is administered as a prophylactic. In such embodiments, the methods of the invention are effective in preventing or protecting a subject from viral infection. In an example embodiment, the composition of the present invention is used as a component of a seasonal and / or pandemic vaccine or as part of a vaccination regimen intended to confer long-lasting (multi-season) protection. In an example embodiment, the composition of the presenting invention is used to treat the symptoms of infection.

[0226] In an example embodiment, the methods of immunizing provided herein elicit a broadly protective immune response against a bacterial infection selected from anthrax, cholera, diphtheria, Haemophilus influenzae type b (Hib), meningitis, pertussis, pneumonia, tetanus, tuberculosis, and typhoid fever.

[0227] In an example embodiment, the methods of immunizing provided herein elicit a broadly neutralizing immune response against a bacterial infection selected from anthrax, cholera, diphtheria, Haemophilus influenzae type b (Hib), meningitis, pertussis, pneumonia, tetanus, tuberculosis, and typhoid fever. In an example embodiment, the immune response comprises an antibody response. Accordingly, in an example embodiment, the composition described herein can offer protection against different types of bacteria.

[0228] In an example embodiment, the composition described herein can offer protection against a bacterial pathogen is selected from a Bacillus, a Bordetella, a Clostridium, a Corynebacterium, a Haemophilus, a Mycobacterium, a Neisseria, a Salmonella, a Streptococcus, and a Vibrio.

[0229] In an example embodiment, the composition described herein can offer protection against a bacterial pathogen selected from B. anthracis, B. pertussis, C. diphtheriae, C. tetani, H. influenzae , M. tuberculosis, N. meningitidis, S. enterica, S. pneumoniae, and V. cholerae.

[0230] In an example embodiment, the subject is a non-human mammal. In an example embodiment, the subject is a farm animal or a pet (e.g., a dog, a cat, a sheep, cattle, and / or a pig). In an example embodiment, the subject is a non-human primate. In an example embodiment, the subject is an avian (e.g., a chicken, a duck, a goose, and / or a turkey).

[0231] In an example embodiment, the subject is a human. In an example embodiment, the subject is an adult, an adolescent, or an infant. In an example embodiment, the human subject is younger than six months of age. In an example embodiment, the human subject is six monthsor older, is six months through 35 months of age, is 36 months through 8 years, or is nine years or older. In an example embodiment, the human subject is an elderly aged 55 years or older, such as 60 or older or 65 years of age or older. Also contemplated by the present invention is the administration of the composition and / or performance of the treatment methods in utero.Processes for Making the Present LP Vaccines

[0232] The present LPs can be prepared using various techniques presently known in art. For example, multilam ellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion that results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0233] Various methods are described in US 2011 / 0244026, US 2016 / 0038432, US 2018 / 0153822, US 2018 / 0125989, and PCT / US2020 / 043223 (filed Jul. 23, 2020) and can be used to practice the present invention. One exemplary process entails encapsulating mRNA by mixing it with a mixture of lipids without first pre-forming the lipids into lipid particles, as described in US 2016 / 0038432. Another exemplary process entails encapsulating mRNA by mixing pre-formed LPs with mRNA, as described in US 2018 / 0153822.

[0234] In an example embodiment, the process of preparing mRNA-loaded LPs includes a step of heating one or more of the solutions to a temperature greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid particles, the solution comprising the mRNA and the mixed solution comprising the LP-encapsulated mRNA. In an example embodiment, the process includes heating one or both of the mRNA and pre-formed LP solutions before the mixing step. In an example embodiment, the process includes heating one or more of the solutions comprising the pre-formed LPs, the solution comprising the mRNA, and the solution comprising the LP-encapsulated mRNA during the mixing step. In an example embodiment, the process includes heating the LP-encapsulated mRNA after the mixing step. In an example embodiment, the temperature to which one or more of the solutions is heated is or is more significant than about 30° C., 37° C., 40° C., 45° C., 50° C., 55° C., 60° C., 65° C., or 70° ° C. In an example embodiment, the temperature to whichone or more of the solutions is heated ranges from about 25-70° C., about 30-70° C., about 35- 70° C., about 40-70° C., about 45-70° ° C., about 50-70° ° C., or about 60-70° C. In an example embodiment, the temperature is about 65° C.

[0235] Various methods may prepare an RNA solution suitable for the present invention. In an example embodiment, RNA may be directly dissolved in a buffer solution described herein. In an example embodiment, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution before mixing with a lipid solution for encapsulation. In an example embodiment, an RNA solution may be generated by mixing an RNA stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In an example embodiment, a suitable RNA stock solution may contain RNA in water or a buffer at a concentration at or greater than about 0.2 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.8 mg / ml, 1.0 mg / ml, 1.2 mg / ml, 1.4 mg / ml, 1.5 mg / ml, or 1.6 mg / ml, 2.0 mg / ml, 2.5 mg / ml, 3.0 mg / ml, 3.5 mg / ml, 4.0 mg / ml, 4.5 mg / ml, or 5.0 mg / ml.

[0236] In an embodiment, an RNA stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps, and centrifugal pumps. Typically, the buffer solution is mixed at a rate greater than that of the RNA stock solution. For example, the buffer solution may be mixed at a rate at least l x, 2x, 3x, 4 / , 5 / , 6x, 7x, 8x, 9 , 10x, 15x, or 20x greater than the rate of the RNA stock solution. In an example embodiment, a buffer solution is mixed at a flow rate ranging between about 100- 6000 ml / minute (e.g., about 100-300 ml / minute, 300-600 ml / minute, 600-1200 ml / minute, 1200-2400 ml / minute, 2400-3600 ml / minute, 3600-4800 ml / minute, 4800-6000 ml / minute, or 60-420 ml / minute). In an example embodiment, a buffer solution is mixed at a flow rate of, or greater than, about 60 ml / minute, 100 ml / minute, 140 ml / minute, 180 ml / minute, 220 ml / minute, 260 ml / minute, 300 ml / minute, 340 ml / minute, 380 ml / minute, 420 ml / minute, 480 ml / minute, 540 ml / minute, 600 ml / minute, 1200 ml / minute, 2400 ml / minute, 3600 ml / minute, 4800 ml / minute, or 6000 ml / minute.

[0237] In an example embodiment, an RNA stock solution is mixed at a flow rate ranging between about 10-600 ml / minute (e.g., about 5-50 ml / minute, about 10-30 ml / minute, about 30-60 ml / minute, about 60-120 ml / minute, about 120-240 ml / minute, about 240-360 ml / minute, about 360-480 ml / minute, or about 480-600 ml / minute). In an example embodiment, an RNA stock solution is mixed at a flow rate of or greater than about 5 ml / minute, 10 ml / minute, 15 ml / minute, 20 ml / minute, 25 ml / minute, 30 ml / minute, 35ml / minute, 40 ml / minute, 45 ml / minute, 50 ml / minute, 60 ml / minute, 80 ml / minute, 100 ml / minute, 200 ml / minute, 300 ml / minute, 400 ml / minute, 500 ml / minute, or 600 ml / minute.

[0238] Incorporating a desired RNA into a lipid particle is referred to as “loading.” Exemplary methods are described in Lasic et a , FEBS Lett. (1992) 312:255-8. The LP- incorporated nucleic acids may be completely or partially located in the lipid particle's interior space, within the lipid particle, within the bilayer membrane of the lipid particle, or associated with the exterior surface of the lipid particle membrane. The incorporation of an RNA into lipid particles is also referred to herein as “encapsulation,” wherein the nucleic acid is entirely or substantially contained within the interior space of the lipid particle.

[0239] Suitable LPs may be made in various sizes. In an example embodiment, decreased lipid particle size is associated with more efficient delivery of an mRNA. Selection of an appropriate LP size may consider the site of the target cell or tissue and, to some extent, the application for which the lipid particle is being made.

[0240] Various methods known in the art are available for sizing a population of lipid particles. Particularly exemplary methods utilize Zetasizer Nano ZS (Malvern Panalytical) to measure LP particle size. In one protocol, 10 pl of an LP sample is mixed with 990 pl of 10% trehalose. This solution is loaded into a cuvette and put into the Zetasizer machine. The z- average diameter (nm), or cumulants mean, is the average size for the LPs in the sample. The Zetasizer machine can also measure the poly dispersity index (PDI) using dynamic light scattering (DLS) and cumulant autocorrelation function analysis. The average LP diameter may be reduced by sonicating the formed LP. Intermittent sonication cycles may be alternated with quasi-elastic light scattering (QELS) assessment to guide efficient lipid particle synthesis.

[0241] In an example embodiment, the majority of purified LPs, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the LPs, have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In an example embodiment, substantially all (e.g., greater than 80 or 90%) of the purified lipid particles have a size of about 70-150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[0242] In an example embodiment, the LPs in the present composition have an average size of less than 150 nm, less than 120 nm, less than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less than 60 nm, less than 50 nm, less than 30 nm, or less than 20 nm.

[0243] In an example embodiment, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the LPs in the present composition have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm) or about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization.

[0244] In an example embodiment, the dispersity, or measure of heterogeneity in the size of molecules (PDI), of LPs in a pharmaceutical composition provided by the present invention is less than about 0.5. In an example embodiment, an LP has a PDI of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.28, less than about 0.25, less than about 0.23, less than about 0.20, less than about 0.18, less than about 0.16, less than about 0.14, less than about 0.12, less than about 0.10, or less than about 0.08. The PDI may be measured by a Zetasizer machine, as described above.

[0245] In an example embodiment, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified LPs in a pharmaceutical composition provided herein encapsulate an mRNA within each particle. In an example embodiment, substantially all (e.g., greater than 80% or 90%) of the purified lipid particles in a pharmaceutical composition encapsulate an mRNA within each particle. In an example embodiment, a lipid particle has an encapsulation efficiency of between 50% and 99%, or greater than about 60, 65, 70, 75, 80, 85, 90, 92, 95, 98, or 99%. Typically, lipid particles used herein have an encapsulation efficiency of at least 90% (e.g., at least 91, 92, 93, 94, or 95%).

[0246] In an example embodiment, an LP has a N / P ratio of between 1 and 10. A lipid particle has a N / P ratio above 1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, or about 8. In an example embodiment, a typical LP has an N / P ratio of 6.

[0247] In an example embodiment, a pharmaceutical composition according to the present invention contains at least about 0.5 pg, 1 pg, 5 pg, 10 pg, 100 pg, 500 pg, or 1000 pg of encapsulated mRNA. In an example embodiment, a pharmaceutical composition contains about 0.1 pg to 1000 pg, at least about 0.5 pg, at least about 0.8 pg, at least about 1 pg, at least about 5 pg, at least about 8 ug, at least about 10 pg, at least about 50 pg, at least about 100 pg, at least about 500 pg, or at least about 1000 pg of encapsulated mRNA.

[0248] In an example embodiment, where the RNA is mRNA, the mRNA can be made by chemical synthesis or by in vitro transcription (IVT) of a DNA template (FIG. 5). In an IVT process, a cDNA template is used to produce an mRNA transcript, and a DNase degrades the DNA template. The transcript is purified by depth and tangential flow filtration (TFF). The purified transcript is further modified by adding a cap and a tail, and the modified RNA is purified again by depth filtration and TFF.

[0249] The mRNA is then prepared in an aqueous buffer and mixed with an amphiphilic solution containing the lipid components of the LPs. An amphiphilic solution for dissolving the four lipid components of the LPs may be an alcohol solution. In an example embodiment, the alcohol is ethanol. The aqueous buffer may be, for example, a citrate, phosphate, acetate, or succinate buffer and may have a pH of about 3.0-7.0, e.g., about 3.5, about 4.0, about 4.5, about 5.0, about 5.5, about 6.0, or about 6.5. The buffer may contain other components such as salt (e.g., sodium, potassium, and / or calcium salts). In an example embodiment, the aqueous buffer has one mM citrate, 150 mM NaCl, pH 4.5.

[0250] An exemplary, nonlimiting process for making an mRNA-LP composition is described in Example 1. The process involves mixing a buffered mRNA solution with a solution of lipids in ethanol in a controlled homogeneous manner, where the ratio of lipids to mRNA is maintained throughout the mixing process. This illustrative example presents the mRNA in an aqueous buffer containing citric acid monohydrate, tri-sodium citrate dihydrate, and sodium chloride. The mRNA solution is added to the solution (1 mM citrate buffer, 150 mM NaCl, pH 4.5). The lipid mixture of four lipids (e.g., a cationic lipid, a PEGylated lipid, a cholesterol-based lipid, and a neutral lipid) is dissolved in ethanol. The aqueous mRNA solution and the ethanol lipid solution are mixed at a volume ratio of 4: 1 in a “T” mixer with a near “pulseless” pump system. The resultant mixture is then subjected to downstream purification and buffer exchange. The buffer exchange may be achieved using dialysis cassettes or a TFF system. TFF may concentrate and buffer-exchange the resulting nascent LP immediately after formation via the T-mix process. The diafiltration process is continuous, keeping the volume constant by adding an appropriate buffer at the same rate as the permeate flow.Packaging of the mRNA-LP Vaccines

[0251] The mRNA-LP vaccines can be packaged for parenteral (e.g., intramuscular, intradermal, or subcutaneous) administration or nasopharyngeal (e.g., intranasal)administration. The vaccine compositions may be an extemporaneous formulation, where the LP composition is lyophilized and reconstituted with a physiological buffer (e.g., PBS) just before use. The vaccine compositions may also be shipped and provided as an aqueous or frozen aqueous solution. They can be directly administered to subjects without reconstitution (after thawing if previously frozen).

[0252] Accordingly, the present disclosure provides an article of manufacture, such as a kit, that provides the mRNA-LP vaccine in a single container or the mRNA-LP vaccine in one container and a physiological buffer for reconstitution in another. The container(s) may contain a single-use dosage or multi-use dosage. The containers may be pre-treated glass vials or ampules. The article of manufacture may also include instructions for use.

[0253] In an example embodiment, the mRNA-LP vaccine is provided for use in intramuscular (IM) injection. The vaccine can be injected into a subject at, e.g., their deltoid muscle in the upper arm. In an example embodiment, the vaccine is provided in a pre-filled syringe or injector (e.g., single-chambered or multi-chambered). In an example embodiment, the vaccine is provided for inhalation and in a pre-filled pump, aerosolizer, or inhaler.

[0254] The mRNA-LP vaccines are administered to subjects in need thereof in a prophylactically effective amount, i.e., an amount that provides sufficient immune protection against a target pathogen for an adequate amount of time (e.g., one year, two years, five years, ten years, or lifetime). Sufficient immune protection may include, for example, preventing or alleviating symptoms associated with infections by the pathogen. In an example embodiment, multiple doses (e.g., two doses) of the vaccine are injected into subjects in need thereof to achieve the desired prophylactic effects. The doses (e.g., prime and booster doses) may be separated by an interval of, e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, one month, two months, three months, four months, five months, six months, one year, two years, five years, or ten years.

[0255] In an example embodiment, a single dose of the mRNA-LP vaccine contains 1-50 pg of mRNA (e.g., monovalent or multivalent). For example, a single dose may contain about 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 12.5 pg, or about 15 pg of the mRNA for intramuscular (IM) injection. In an example embodiment, a multi -valent single dose of an LP vaccine contains multiple (e.g., 2, 3, or 4) kinds of LPs, each for a different antigen. Each type of LP has an mRNA amount of, e.g., 2.5 pg, about 5 pg, about 7.5 pg, about 10 pg, about 12.5 pg, or about 15 pg.Kits

[0256] The present invention also provides a kit, or a kit of parts, comprising the composition and / or the vaccine according to the invention, optionally a liquid vehicle for solubilizing and optionally technical instructions with information on the administration and dosage of the mRNA sequence, the composition and / or the vaccine. The technical instructions may contain information about the administration and dosage of the composition and / or the vaccine. Such kits, or kits of parts, may be applied, e.g., for any of the applications mentioned above or uses, such as for the preparation of an inventive medicament, e.g., a vaccine, for the treatment or prevention of an infectious disease or disorder disclosed herein. The kits may also be applied for the use of the composition or the vaccine as defined herein (for the preparation of an inventive vaccine) for the treatment or prevention of the infection, disease, or disorder disclosed herein; the composition and / or the vaccine may be capable of inducing or enhancing an immune response in a mammal as defined above. Such kits may further be applied for the use of the composition or the vaccine as described herein (for the preparation of an inventive vaccine) for modulating, preferably for eliciting, e.g., to induce or enhance, an immune response in a mammal as defined above, and preferably for supporting treatment or prevention of the infection or disease or disorder disclosed herein. Kits of parts, as a special form of kits, may contain one or more identical or different compositions and / or one or more identical or different vaccines as described herein in other parts of the kit. Kits of parts may also contain an (e.g., one) composition, an (e.g., one) vaccine according to the invention in different parts of the kit. The above kits may be used in treatment or prevention.

[0257] According to the present invention, the kit may contain at least one adjuvant. In an example embodiment, the kit may contain at least one further pharmaceutically active component, preferably a therapeutic compound suitable for the treatment and / or prevention of cancer or a related disorder. Moreover, in an example embodiment, the kit may additionally contain parts and / or devices necessary or suitable for administering the composition or the vaccine according to the invention, including needles, applicators, patches, and injection devices.General Definitions

[0258] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found inMolecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (FM. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995) (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton etal., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0259] As used herein, the singular forms “a,” “an,” and “the” include both singular and plural referents unless the context dictates otherwise.

[0260] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated, having, for example, from one to twenty- four carbon atoms (C1-C24 alkyl), six to twenty-four carbon atoms (C6-C24 alkyl), four to twenty carbon atoms (C4-C20 alkyl), six to sixteen carbon atoms (Ce-Ci6 alkyl), six to nine carbon atoms (C6-C9 alkyl), one to fifteen carbon atoms (C1-C15 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (Ci-Cs alkyl) or one to six carbon atoms (Ci-Ce alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n-propyl, 1 methylethyl (iso propyl), n-butyl, n-pentyl, 1,1 -dimethylethyl (t butyl), 3 -methylhexyl, 2- methylhexyl, and the like. Unless stated otherwise in the specification, an alkyl group is optionally substituted.

[0261] “Alkenyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds, and having, for example, from two to twenty-four carbon atoms (C2-C24 alkenyl), six to twenty- four carbon atoms (C6-C24 alkenyl), four to twenty carbon atoms (C4-C20 alkenyl), six to sixteen carbon atoms (Ce-Ci6 alkenyl), six to nine carbon atoms (C6-C9 alkenyl), two to fifteen carbonatoms (C2-C15 alkenyl), two to twelve carbon atoms (C2-C12 alkenyl), two to eight carbon atoms (C2-C8 alkenyl) or two to six carbon atoms (C2-C6 alkenyl) and which is attached to the rest of the molecule by a single bond, e.g., ethenyl, prop-l-enyl, but-l-enyl, pent-l-enyl, penta-1,4- dienyl, and the like. Unless stated otherwise in the specification, an alkenyl group is optionally substituted.

[0262] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated and having, for example, from one to twenty-four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene), one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (Ci-Cs alkylene), one to six carbon atoms (Ci-Ce alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the radical group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and the radical group can be through one carbon or two carbons within the chain. Unless stated otherwise in the specification, an alkylene chain may be optionally substituted.

[0263] The term “substituted” means at least one hydrogen atom is replaced by a bond to a non-hydrogen atom such as, but not limited to: a halogen atom such as F, Cl, Br, or I; oxo groups (=0); hydroxyl groups ( — OH); C1-C12 alkyl groups; cycloalkyl groups; — (C=O)OR'; — O(C=O)R'; — C(=O)R'; —OR'; — S(O)XR'; — S— SR'; — C(=O)SR'; — SC(=O)R'; — NR'R'; — NR'C(=0)R'; — C(=0)NR'R'; — NR'C(=0)NR'R'; — 0C(=0)NR'R'; — NR'C(=0)0R'; — NRS(O)XNR'R'; — NR'S(O)XR'; and — S(O)XNR'R', wherein: R' is, at each occurrence, independently H, C1-C15 alkyl or cycloalkyl, and x is 0, 1 or 2. In an example embodiment, the substituent is a C1-C12 alkyl group. In an example embodiment, the substituent is a cycloalkyl group. In an example embodiment, the substituent is a halo group, such as fluoro. In an example embodiment, the substituent is an oxo group. In an example embodiment, the substituent is a hydroxyl group. In an example embodiment, the substituent is an alkoxy group ( — OR'). In an example embodiment, the substituent is a carboxyl group. In an example embodiment, the substituent is an amino group ( — NR'R').

[0264] The term “optional” or “optionally” means that the subsequently described event, circumstance, or substituent may or may not occur. The description includes instances where the event or circumstance occurs and instances where it does not.

[0265] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges and the recited endpoints.

[0266] The terms “about” or “approximately,” as used herein when referring to a measurable value, such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less, + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is also specifically and preferably disclosed.

[0267] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid.” The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example, by puncture or other collecting or sampling procedures.

[0268] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells, and the progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0269] “Pharmaceutically acceptable salt” includes both acid and base addition salts.

[0270] “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor- 10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfuric acid, ethane- 1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxy ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-l,5-disulfonic acid, naphthalene-2-sulfonic acid, l-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid, and the like.

[0271] “Pharmaceutically acceptable base addition salt” refers to salts that retain the free acids' biological effectiveness and properties, which are not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include but are not limited to sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, etc. Ammonium, sodium, potassium, calcium, and magnesium salts are preferred as inorganic salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2- dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0272] A “pharmaceutical composition” refers to a formulation of a compound of structure (I) and a medium generally accepted in the art for delivering the biologically active compound to mammals, e.g., humans. Such a medium includes all pharmaceutically acceptable carriers, diluents or excipients therefor.

[0273] “Effective amount” or “therapeutically effective amount” refers to that amount of a compound of structure (I) which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a lipid particle of embodiments the invention which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his knowledge and to this disclosure.“Treating” or “treatment” as used herein covers the treatment of the disease or condition of interest in a mammal, preferably a human, having the disease or condition of interest, and includes: (i) preventing the disease or condition from occurring in a mammal, in particular, when such mammal is predisposed to the condition but has not yet been diagnosed as having it; (ii)inhibiting the disease or condition, i.e., arresting its development; (iii) relieving the disease or condition, i.e., causing regression of the disease or condition; or (iv) relieving the symptoms resulting from the disease or condition, i.e., relieving pain without addressing the underlying disease or condition. As used herein, the terms “disease” and “condition” may be used interchangeably or may be different in that the particular disorder or condition may not have a known causative agent (so that etiology has not yet been worked out). It is, therefore, not yet recognized as a disease but only as an undesirable condition or syndrome wherein clinicians have identified a more or less specific set of symptoms.

[0274] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.

[0275] The following Examples illustrate further embodiments. They are given for illustrative purposes only and are not intended to limit the scope of the invention.EXAMPLESExample 1 - Preparation of an Exemplary Lipid Particle Composition

[0276] An exemplary LP composition was prepared, as shown in Table 1. Briefly, Compound 1, DSPC, cholesterol, and DMG-PEG2000 were solubilized in ethanol at a molar ratio of approximately 50: 10:38.5: 1.5. Syringe pumps were used to mix the ethanolic lipid solution with the mRNA aqueous solution at a ratio of about 1 :3 (vol / vol) with total flow ratesabove 15 ml / min. The ethanol was then removed, and the external buffer was replaced with PBS by dialysis. Finally, the lipid nanoparticles were filtered through a 0.2 m pore sterile filter. Lipid particle size was 95-140 nm diameter as determined by quasi-elastic light scattering using a Malvern Zetasizer Nano (Malvern, UK).Table 1. Exemplary LP FormulationTotal Prep Volume Flow Rate Ratio N / P # ionizable amines mL (aq:organic)Example 2 - mRNA / LP Characterization

[0277] Tables 3-5 provide information on the dose size, zeta potential, and poly dispersity index (PI) of the mRNA / LP formulation described in Example 1.First Second Zeta Zeta Potential PI 1st PI 2ndDose Dose Size Potential 1st 2nd Dose Dose DoseSize* DoseCCHFV- 95.79 114.4 -7.2 -3 0.019 0.111UTRT-UTR 104.6 139.3 -4.6 -0.34 0.009 0.08 ™Table 5. Second Dose2 T-UTR 86.6 951.9 824.0Example 3 - Determination of PkA of Formulated Lipids

[0278] The pKa of formulated lipids is correlated with the effectiveness of LPs for the delivery of nucleic acids (see Jayaraman et al., Angewandte Chemie, International Edition (2012), 51(34), 8529-8533; Semple et al., Nature Biotechnology 28, 172-176 (2010)). In an example embodiment, the preferred range of pKa is ~5 to ~7. The pKaof representative lipids was determined in lipid particles using an assay based on fluorescence of 2-(p-toluidino)-6- napthalene sulfonic acid (TNS). mRNA / LP formulation described in Example 1 in PBS at a 0.4 mM total lipid concentration was prepared. TNS was prepared as a 100 pM stock solution in distilled water. Vesicles were diluted to 24 pM lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, and 130 mM NaCl, where the pH ranged from 2.5 to 11. An aliquot of the TNS solution was added to give a final concentration of 1 pM and following vortex mixing, fluorescence intensity was measured at room temperature in a SLM Aminco Series 2 Luminescence Spectrophotometer using excitation and emission wavelengths of 321 nm and 445 nm. A sigmoidal best-fit analysis was applied to the fluorescence data, and the pKawas measured as the pH, giving rise to half-maximal fluorescence intensity, as shown in FIGS. 8A-8C.Example 4 - Optimizing mRNA-lipid nanoparticle architecture for protective immunity against Crimean-Congo hemorrhagic fever virus in C57BL / 6 mice

[0279] Applicants report the rational design and optimization of mRNA-lipid nanoparticle (LNP) vaccine candidates targeting Crimean-Congo hemorrhagic fever virus (CCHFV) (see FIG. 37). To accomplish this, Applicants evaluated the immunogenicity of nucleoprotein (N), glycoprotein GP85, and their modified variants by testing three different untranslated region (UTR) configurations, three distinct 5' cap structures, and a lipid nanoparticle formulation incorporating a novel ionizable lipid, BP- 104. By systematically evaluating these parameters, the disclosed approach maximizes antigen expression and elicits potent innate and adaptive immune responses in C57BL / 6 mice. These results establish a next-generation CCHFV mRNA-LNP vaccine platform.Results-Antigen Selection, mRNA Generation, Expression, and Characterization

[0280] Applicants selected nucleocapsid (NCBI Reference Sequence: NP 950237.1) and GP85 (GenBank: QBW95916.1) as the primary immunogens. The nucleocapsid has been shown to activate both T cell and B cell responses when delivered via various vaccine platforms, as reported in prior studies. GP38 has also demonstrated promising immunogenicity in mouse models, stimulating antibody responses that confer protective effects. In the present disclosure, Applicants incorporated a mucin-like domain into the GP38-based vaccine candidates to generate GP85, as this domain is essential for maintaining GP38's correct structural conformation.

[0281] In addition to codon-optimized wild-type versions of these two antigens, Applicants generated a modified form of nucleocapsid in which the DEVD motif — responsible for caspase 3 cleavage — was substituted with AEVA, and a modified GP85 in which two N-glycosylation sites were removed from the GP38 sequence, designated as GP85 (GP38Aglyc). All codon- optimized sequences were cloned into pcDNA™3.1 (+) and subsequently transferred into the pUC57-PolyA vector containing a segmented poly(A) tail. The constructs further incorporated desired untranslated regions (UTRs), a Kozak sequence, start and three stop codons, and a T7 promoter. These plasmids were linearized and used as templates for mRNA synthesis (see FIGS. 39A, 41A, and 42A). The mRNAs were capped using Anti-Reverse Cap Analog (ARCA), adenosine-guanosine (AG) cap, or CleanCap M6 for subsequent in vivo mouse immunization experiments.

[0282] To evaluate the influence of UTRs on the immunogenicity of mRNA / LNP constructs, Applicants generated three different UTR variants: CCHFV (derived from the S segment of CCHFV), T (as previously described in related studies), and ACTA-1(corresponding to the UTR region of the ACTA-1 gene, encoding skeletal muscle alpha-actin, the predominant actin isoform in adult skeletal muscle) within the plasmid containing wildtype nucleocapsid (see FIG. 39A). The predicted secondary RNA structures of these UTRs are shown in FIG. 39 A. The ARCA-capped mRNAs were synthesized incorporating Nl- methylpseudouridine-5 '-triphosphate and verified by native agarose gel electrophoresis (FIG. 39B) and by protein expression analysis in HEK293T cells 18 hours post-transfection.

[0283] For additional in vivo studies, Applicants prepared mRNA constructs encoding nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc) incorporating AG or CleanCap M6 caps and modified nucleotides Nl-methylpseudouridine-5 '-triphosphate and 5-methylcytidine-5'- triphosphate (see FIGS. 41 A and 42 A). The mRNAs were verified by agarose gel electrophoresis (FIGS. 41B and 42B). CleanCap M6 mRNAs were transfected into HEK293T, HepG2, and C2C12 cells, and expression of the encoded proteins was confirmed by western blot analysis 18 hours post-transfection. Nucleocapsid (N) and Nmut were detected at approximately 54 kDa, and both GP38 and GP38Aglyc derived from GP85 mRNAs were detected at 38 kDa as expected (see FIG. 42F).Preparation and characterization of mRNA-LNPs

[0284] mRNA-lipid nanoparticles (mRNA-LNPs) were formulated using a microfluidic mixing device, in which an ethanolic lipid solution was combined with an aqueous mRNA phase under controlled flow conditions. A novel ionizable lipid designated BP- 104 (see FIG. 38 A) was employed, and the lipid mixture included BP- 104, DMG-PEG2000, DSPC, and cholesterol according to the formulation composition shown in FIG. 38B. The resulting mRNA / LNPs were buffer-exchanged, concentrated, and stored at -80°C. Dynamic light scattering (DLS) analysis demonstrated a monodisperse particle size distribution for all ARCA- , AG-, and M6-capped mRNA constructs, with poly dispersity index (PDI) values below 0.15 for each formulation (see FIGS. 39C, 41C, and 42C). The LNPs exhibited near-neutral zeta potential (-8 ± 1 mV) and greater than 90% encapsulation efficiency, consistent across multiple mRNA cargos (data not shown).

[0285] The apparent pKa of the mRNA / LNPs was determined using a 2-(p- toluidinyl)naphthalene-6-sulfonic acid (TNS) binding assay, yielding values of 6.94 for nucleocapsid, 6.82 for Nmut, 6.73 for GP85, and 6.62 for GP85 (GP38Aglyc) in CleanCap M6 mRNA formulations (see FIG. 42E). The CleanCap M6 mRNA / LNPs were added to HEK293T cells, and proliferation assays conducted 24 hours post-treatment indicated that only theNmut / LNP construct exhibited no measurable cytotoxicity and maintained proliferation rates comparable to the negative control (l x PBS). In contrast, the ionizable LNP control (iLNP) showed mild cytotoxicity compared with the negative control, and among all vaccine constructs, the nucleocapsid formulation exhibited the highest cytotoxicity in vitro (see FIG. 42D).Recombinant protein production and purification

[0286] To design immunological assays to evaluate the immunogenicity of the disclosed mRNA / LNP constructs in the mouse model, recombinant nucleocapsid and GP38 proteins were produced and purified. For bacterial expression of nucleocapsid, a plasmid designated pET30-N was constructed, incorporating a 6xHis tag at the C-terminus. Additional constructs were prepared in the same vector by introducing a Strep-Tag II sequence at either the N- or C- terminus; however, these constructs yielded lower protein quantities and purities than the His- tag version. Accordingly, the His-tag construct was selected for subsequent experiments. After sequence confirmation, the plasmid was transformed into E. coli Rosetta cells, which exhibited superior protein production relative to BL21 and Rosetta 2 strains. Comparable protein yield and purity were observed when bacterial induction was performed at 16 °C for 18 hours, 29 °C for 5 hours, or 37 °C for 3 hours; therefore, induction at 37 °C for 3 hours was adopted. Following Ni-NTA purification by gravity-flow chromatography, nucleocapsid protein was recovered in the 250 mM imidazole elution fraction.

[0287] Recombinant GP38 protein was produced in ExpiCHO cells and compared to expression in Freestyle 293F cells; ExpiCHO cultures yielded substantially higher GP38 protein levels under identical conditions. A furin-expressing plasmid was co-transfected to promote cleavage of GP38 from its mucin-like domain at the endogenous furin cleavage site. The GP85 expression construct contained both a 6xHis tag and a Strep-Tag II at the C- terminus, and GP38 was purified using Strep-Tactin affinity chromatography. Elution fractions 2 through 5 were pooled, concentrated, and stored at -80 °C.

[0288] For size-exclusion chromatography (SEC) of the nucleocapsid, multiple column and buffer conditions were evaluated owing to the protein's RNA-binding properties and instability. Optimal resolution was achieved using a buffer comprising 20 mM Tris-HCl, 300 mM NaCl, and 10 % glycerol with a Superdex 75 Increase 10 / 300 GL column. For GP38, phosphate-buffered saline (PBS) containing 10 % glycerol was used as the SEC buffer. Following SEC purification, distinct bands corresponding to 54 kDa for nucleocapsid (FIG.40A) and 38 kDa for GP38 (FIG. 40B) were observed by Coomassie blue staining. The purified fractions were pooled, concentrated using 50 kDa Amicon filters, snap-frozen, and stored at - 80 °C.Comparing in vitro Immunogenicity of BP-104 ionizable Lipid with SM-102 and ALC- 0315 encapsulating Nmut

[0289] The potential of the disclosed lipid nanoparticle (LNP) formulation to stimulate innate immune responses was compared in vitro with LNPs generated using SM-102 and ALC- 0315, two ionizable lipids employed in FDA-approved mRNA vaccine formulations (see FIG. 38 A). The Nmut mRNA construct was selected for encapsulation, as it exhibited the lowest cytotoxicity in cell culture based on proliferation assays. As shown in FIG. 38C, all three mRNA / LNP constructs stimulated type I interferon (IFN-I) and interleukin-6 (IL-6) production after 24 hours in IFN-a / p and IL-6 reporter HEK 293 cells, respectively. The results are expressed as fold changes relative to the negative control. No significant differences were observed among the mRNA / LNP groups except at concentrations of 1 pg and 62.5 ng, where the Nmut-ALC0315 formulation exhibited a significantly higher IFN-I fold-change compared to the BP- 104-based LNP carrying Nmut (see FIG. 38C). All three empty LNPs (iLNP-BP104, iLNP-SM102, and iLNP-ALC0315) also stimulated measurable IFN-1 and IL-6 responses, demonstrating intrinsic adjuvant potential (see FIG. 38C).

[0290] The same assay was performed using THPl-Dual™ cells to evaluate NF-KB signaling pathway activation as a marker of innate immune activation. Consistent with the IFN- I and IL-6 data, all mRNA / LNPs and iLNPs stimulated NF-KB pathway activation (see FIG. 38C). In the antigen cross-presentation assay (see FIG. 38D), all three mRNA / LNP formulations induced production of IFN-y, TNF-a, and IL-17A cytokines in CD4+cells after four days of co-culture (see FIG. 38E). Among these, the ALC0315-Nmut formulation produced significantly higher levels of all three cytokines compared to other LNP formulations. Cytokine and chemokine levels were also quantified from cell culture supernatants collected four days after co-culture. No significant differences were observed among the formulations, except for MCP- 1 , for which ALC0315-Nmut induced a significantly higher concentration than the other groups (see FIG. 38F). mRNA / LNP Constructs Stimulated Robust Cytokines in Splenic T Cells

[0291] To evaluate in vivo immunogenicity of the generated mRNA / LNP constructs, splenocytes were collected on day 28 for ARCA- and AG-capped formulations and on day 32for CleanCap M6-capped formulations. The collected cells were analyzed by flow cytometry for intracellular cytokine expression and by ELISPOT for cytokine secretion following 24 hours of in vitro stimulation with the corresponding antigen (5 pg / mL).

[0292] In the ARCA-capped experiment (see FIG. 39D), all three vaccine groups elicited robust intracellular IFN-y, IL-2, and TNF-a production in both CD4+and CD8+T cells. In addition, the ACTA-l-UTR group demonstrated significantly higher IL-4 and IL-17A responses in CD4+cells (see FIG. 39G). The T-UTR group exhibited significantly higher responses in CD8+cells secreting all three cytokines (see FIG. 39F). In the ELISPOT assay, all three constructs induced significantly elevated IL-2 secretion with no difference among groups, whereas ACTA-1- and CCHFV-UTR constructs showed significantly higher IFN-y responses compared to the T-UTR and negative-control groups (see FIG. 39E).

[0293] In the AG-capped experiment (see FIG. 4 IE), cytokine secretion from splenic T cells was analyzed by ELISPOT. The nucleocapsid and Nmut vaccine groups elicited significantly higher IFN-y responses than the other vaccine and control groups. Conversely, GP85 and GP85 (GP38Aglyc) formulations induced significantly higher IL-4 responses, demonstrating a Th2-biased immunostimulatory profile relative to the other vaccine candidates.

[0294] In the CleanCap M6-capped experiment (see FIG. 42G), four analogous mRNA / LNP formulations were evaluated in C57BL / 6 mice under a booster immunization regimen. Splenocytes collected post-immunization showed that total CD4+and CD8+T cell frequencies in all vaccine groups were elevated relative to the iLNP negative-control group (see FIG. 42K). Among CD4+cells, only the GP85 (GP38Aglyc) formulation produced significantly higher IL-2 and TNF-a levels compared to the negative control, with no significant differences among vaccine groups in CD4+cells secreting IFN-y, IL-2, or TNF-a. In contrast, nucleocapsid-immunized mice exhibited significantly enhanced IFN-y and TNF-a responses in CD8+cells relative to the negative-control group (see FIG. 42K).Immunization with AG-Capped mRNA-LNPs expands Tfh and Antigen-Specific GC B cells

[0295] T follicular helper (Tfh) cells play a central role in orchestrating humoral immune responses by supporting germinal center (GC) formation, promoting immunoglobulin class switching, facilitating affinity maturation, and enabling the generation of long-lived memory B cells. To evaluate these responses, inguinal lymph nodes were collected from AG-cappedmRNA / LNP-immunized mice two weeks after the booster dose and analyzed by flow cytometry. As shown in FIG. 4 IF, nucleocapsid, and Nmut formulations stimulated significantly higher frequencies of Tfh cells than GP85, GP85 (GP38Aglyc), and the negative control groups, with Nmut exhibiting the strongest response. Similar to the Tfh cell profile, nucleocapsid and Nmut also induced significantly higher GC B cell responses relative to the negative control group. GP85 (GP38Aglyc) generated a higher GC B cell response than GP85, although the difference was not statistically significant. Antigen-specific GC B cell populations were also quantified, and as shown in FIG. 4 IF, all vaccine groups demonstrated significantly elevated antigen-specific GC B cell frequencies compared to the negative control group.Different Capped mRNA / LNPs Affect Humoral Responses in Mice

[0296] Serum samples were collected 2 weeks after booster administration for the ARCA- and AG-capped mRNA / LNP groups and 3 weeks after booster administration for the CleanCap M6-capped groups to evaluate binding antibody responses using an in-house ELISA assay. In the ARCA-capped mRNA / LNP experiment, no detectable antibody responses were observed in C57BL / 6 mice immunized with nucleocapsid constructs containing different UTR configurations. Therefore, additional immunizations were performed in BALB / c mice using ARCA-capped nucleocapsid and Nmut mRNA / LNP formulations. Positive antibody responses were detected in three of four mice in both vaccine groups at a 1 :50 serum dilution (see FIG. 39H).

[0297] In the AG-capped mRNA / LNP experiment (see FIG. 41G), serum samples collected from immunized C57BL / 6 mice showed two total IgG-positive samples for Nmut and none for the nucleocapsid group. In contrast, both GP85 and GP85 (GP38Aglyc) formulations elicited robust, statistically significant antibody responses compared with the negative control.

[0298] In the CleanCap M6-capped mRNA / LNP experiment (see FIG. 421), stronger antibody responses were observed for Nmut (three positive samples out of six) and significantly higher total IgG levels for GP85 and GP85 (GP38Aglyc) groups, consistent with the results obtained in the AG-capped experiment. No neutralizing antibodies were detected in either the GP85- or GP85 (GP38Aglyc)-immunized mice when tested using pseudotyped particles expressing the modified M protein under both AG and CleanCap M6 conditions.Dendritic Cells Measurement in Lymph Nodes of CleanCap M6 Experiment

[0299] Plasmacytoid dendritic cell (pDC) and conventional dendritic cell type 1 (cDCl) populations were analyzed in the lymph nodes of mice immunized with CleanCap M6-capped mRNA / LNP formulations. As shown in FIG. 42J, the GP85 formulation induced a significantly elevated pDC population compared to GP85 (GP38Aglyc) and the negative control group, whereas the GP85 (GP38Aglyc) group showed no detectable pDCs. In contrast, the nucleocapsid-immunized group exhibited a significantly higher cDCl population than the negative control, and other vaccine groups also showed relatively higher cDCl frequencies than negative control animals.Discussion

[0300] Applicants systematically engineered and evaluated mRNA-lipid nanoparticle (LNP) vaccine candidates targeting Crimean-Congo hemorrhagic fever virus (CCHFV). Using a modular design framework (see FIG. 37), critical regulatory elements — including cap structures, untranslated regions (UTRs), and ionizable lipid chemistries — were interrogated to delineate their effects on translation, innate immune sensing, adaptive immunogenicity in preclinical mouse models.

[0301] CCHFV nucleoprotein (N) and GP85 (mucin-GP38) were selected as the principal antigens based on their high immunological relevance. The nucleocapsid is abundantly expressed and highly conserved, while GP38 is a secreted glycoprotein that elicits nonneutralizing antibodies. Four antigenic constructs were designed: N, a modified N (Nmut), GP85, and a deglycosylated GP38 variant of GP85 (GP38Aglyc). The Nmut construct incorporated an AEVA motif in place of the native caspase-3 cleavage site (DEVD, residues 266-269) to assess whether disruption of the cleavage site could shift antigen processing pathways.

[0302] Regulatory sequence optimization demonstrated distinct UTR-dependent effects. Three UTR configurations — CCHFV-derived, a previously reported sequence (T-UTR), and ACTA-1 — were evaluated using nucleocapsid as the encoded antigen. Both CCHFV and ACTA-1 UTRs enhanced T-cell responses relative to the T-UTR, as determined by intracellular cytokine staining and ELISPOT analysis, leading to selection of the viral UTR for subsequent vaccine candidate development.

[0303] The capping strategy further modulated immune responses in the mouse model. Notably, cap structure influenced B-cell immunogenicity of Nmut: among ARCA, AG, and CleanCap M6 configurations, AG and M6 produced measurable IgG responses in C57BL / 6mice, with M6 yielding the highest titers, whereas ARCA constructs failed to elicit detectable antibody responses in this strain. AG capping consistently produced stronger ELISPOT responses, particularly for N and Nmut, while CleanCap M6 induced cytokine responses comparable to or slightly lower than those with AG for GP38 antigens, illustrating the effect of cap chemistry on Thl- and Th2-biased immunity when mRNAs encode identical antigens.

[0304] LNP composition was optimized by comparing a novel ionizable lipid, BP- 104, with clinically utilized SM-102 (Modema) and ALC-0315 (Pfizer-BioNTech) in the delivery of Nmut mRNA (see FIG. 38). All three ionizable lipids, in both empty and encapsulated forms, supported efficient mRNA delivery and activation of innate immune pathways, including type I interferon (IFN-I) and NF-KB signaling in vitro. However, ALC-0315 formulations induced higher IL-17A and TNF-a production in dendritic cell-naive CD4+T-cell co-cultures, highlighting the immunomodulatory influence of lipid chemistry.

[0305] Comprehensive in vivo testing of the final candidates — N, Nmut, GP85, and GP85 (GP38Aglyc) — encapsulated with CCHFV UTRs and AG caps in BP- 104 LNPs demonstrated potent immunogenicity. N and Nmut induced robust IFN-y responses by both CD4+and CD8+T cells, whereas IL-4 was preferentially elicited by GP85 antigens. T follicular helper (Tfh) cell frequencies were highest in the Nmut group, and germinal center (GC) B cells were enriched in N and Nmut recipients. As expected, GP85 and GP85 (GP38Aglyc) generated strong antibody titers; notably, total IgG responses were also detected in the Nmut group, correlating with elevated Tfh and GC B cell presence. CleanCap M6 constructs yielded similar T cell responses but attenuated IL-4 production and reduced IFN-y for GP85 constructs. Nmut consistently outperformed N across cap configurations in antibody stimulation, establishing it as the preferred nucleocapsid-based candidate; both showed similar cytokine responses in subsequent efforts. However, further studies are needed to improve the durability and effectiveness of this antibody response.

[0306] Collectively, all N and Nmut mRNA-LNP formulations with different UTRs and cap structures induced robust antigen-specific T cell immunity. On the other hand, GP85 and GP85 (GP38Aglyc) elicited highly significant antibody responses, regardless of cap structure. N-based constructs skewed towards Thl-polarized responses (IFN-y, TNF-a), while GP85 constructs promoted a Th2-biased profile with enhanced humoral immunity, validating N / Nmut and GP85 as complementary antigens for CCHFV vaccine development. ARCA capping was suboptimal for antibody induction for N and Nmut in C57BL / 6 mice. In contrast,AG and M6 caps supported the generation of humoral responses in Nmut in the same mice strain, with AG conferring superior cellular responses. Deglycosylated GP38 retained immunogenicity but in a lower amount when compared to naive GP85, suggesting that glycan removal does not significantly impair antigen recognition and may improve epitope exposure.

[0307] Analysis of draining lymph nodes provided mechanistic insight: nucleocapsid (N) and Nmut preferentially expanded conventional dendritic cells (cDCl), which are critical for CD8+T cell priming, whereas GP85 antigens elicited stronger plasmacytoid dendritic cell responses, potentially underpinning their superior antiviral potential through interferon (IFN) production.

[0308] These findings establish a preclinical framework for the optimized design of a CCHFV mRNA-LNP vaccine. Incorporating viral UTRs, 5’ capping for GP85 and Nmut, and rationally selected ionizable lipids resulted in potent immunogenicity for all constructs and protectivity for GP85 and GP85 (GP38Aglyc).MethodsStudy Design

[0309] This study systematically evaluated the immunogenicity and translational efficiency of mRNA-lipid nanoparticle (LNP) formulations targeting Crimean-Congo hemorrhagic fever virus (CCHFV) antigens, focusing on untranslated regions (UTRs), cap structures, and ionizable lipid compositions (FIG. 37). A series of in vitro and in vivo experiments were conducted in a stepwise manner to assess each variable. In the first phase, mRNA constructs encoding the CCHFV nucleocapsid (N) protein were synthesized using three different 5' and 3' UTR pair combinations to test their effect on translation efficiency. All constructs were capped using the ARCA (CapO) system. In the second phase, mRNAs encoding wild-type N, a mutant form of nucleocapsid (Nmut), GP85, and GP85 containing deglycosylated GP38 (GP38Aglyc) were synthesized using AG CleanCap (Capl) and CCHFV- originated UTRs. In the third phase, the same antigen targets (N, Nmut, GP85, and GP85 (GP38Aglyc)) were transcribed using CleanCap M6 (Capl) to assess the impact of additional methylation on cap structure, translation, and immunogenicity. In addition, the role of ionizable lipid chemistry in delivery and immune activation was evaluated. The LNPs were formulated using a novel ionizable lipid (BP- 104). Prior to conducting in vivo experiments in C57BL / 6 mice, the immunogenicity potential of the ionizable lipid BP- 104 was compared with twoclinically used ionizable lipids — SM-102 (Moderna) and ALC-0315 (BioNTech / Pfizer) — in vitro, all of which encapsulate the mutant form of the nucleocapsid (Nmut).Cells

[0310] Four cell lines were used to assess mRNA-LNP construct expression, cellular processing, and recombinant protein production: HEK293T, C2C12, HepG2, and ExpiCHO- S. HEK293T cells (human embryonic kidney cells expressing SV40 large T-antigen; ATCC CRL-11268) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% penicillin-streptomycin (Gibco), and maintained at 37°C in a humidified 5% CO2 incubator. C2C12 cells (mouse myoblast cell line; ATCC CRL-1772) were cultured in DMEM with 10% FBS and 1% penicillin-streptomycin at 37°C with 5% CO2. HepG2 cells (human hepatocellular carcinoma cell line; ATCC HB-8065) were cultured in Minimum Essential Medium (MEM) (Gibco) supplemented with 10% FBS, 1% non-essential amino acids (NEAA), and 1% penicillin- streptomycin. Cells were maintained at 37°C and 5% CO2. ExpiCHO-S cells (Thermo Fisher Scientific, Cat# A29127) were maintained in ExpiCHO™ Expression Medium (Thermo Fisher Scientific) according to the manufacturer's protocol. These cells were cultured in Erlenmeyer flasks in a shaking incubator at 130 rpm, 37°C, 8% CO2, and passaged every 3-4 days. ExpiCHO cells were used for purification of recombinant GP38 protein.

[0311] In addition, HEK-Blue IFN-a / p (InvivoGen, Cat# hkb-ifnabv2), THPl-Dual and HEK-Blue IL-6 (InvivoGen, Cat number: hkb-hil6) cell lines were employed to evaluate type I interferon activity and innate immune pathway activation in response to Nmut mRNA-LNPs formulated with BP104, SM-102, or ALC-0315. HEK-Blue IFN-a / p and HEK-Blue IL-6 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U / mL penicillin, 100 pg / mL streptomycin, 100 pg / mL Normocin, 30 pg / mL Blasticidin, and 100 pg / mL Zeocin. Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were passaged upon reaching 70-80% confluency using phosphate-buffered saline (lx PBS) at 37 °C for 2-3 minutes to detach adherent cells. THPl-Dual cells (InvivoGen, Cat# thpd-nfis) were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U / mL penicillin, 100 pg / mL streptomycin, 100 pg / mL Normocin, 10 pg / mL Blasticidin, and 100 pg / mL Zeocin. Cultures were maintained at 37 °C in a humidified atmosphere with 5% CO2. THPl-Dual cells grow in suspension and were passaged every 2-3 days to maintain a cell density between 2 * 105and 1 x 106cells / mL.

[0312] All cell lines were routinely tested for mycoplasma contamination and used within 15 passages after revival from authenticated stocks.Plasmids

[0313] The human codon-optimized nucleocapsid (N) and GP85 sequences were synthesized by GenScript and received in a pUC57-based plasmid. These genes were subsequently inserted into the pCDNA3.1 plasmid using Gibson Assembly to generate pGSCD-N and pGSCD-Mucin-GP38. A modified version of N was generated by replacing the DEVD motif with AEVA in the wild-type sequence, and a deglycosylated GP38 variant of the GP85 plasmid was constructed by substituting N-X-S / T with Q-X-S / T in the GP38 sequence. The N-linked glycosylation sites were confirmed using the NetNGlyc online tool (available at services. healthtech. dtu.dk / services / NetNGlyc-1.0 / ). The mucin sequence itself was not modified. These four constructs were used as templates to generate plasmids for mRNA synthesis.

[0314] To create expression plasmids, the pGSCD-Mucin-GP38 construct was engineered to include a 6><His tag and a Strep Tag II at the 3' region of GP38 before the stop codon. In parallel, a bacterial codon-optimized nucleocapsid sequence (obtained from GenScript) was inserted into pET-30a(+) (Novagen, catalog no. 69909-3) to produce the pET30-GSCD-N plasmid for bacterial protein purification. In addition, a pUC57-PolyA plasmid containing a segmented poly(A) tail was synthesized by GenScript, as described elsewhere, to allow insertion of a T7 promoter, untranslated regions (5' and 3' UTRs), Kozak sequence, and antigen-coding regions followed by three stop codons, thereby generating a DNA template for in vitro transcription of mRNA.Recombinant Protein Purification

[0315] N protein was produced in the bacterial system as previously described. Briefly, the pET30-N plasmid was transformed into Rosetta bacteria, and a single colony was inoculated in 20 ml of terrific broth (TB) supplemented with 25 pg / pl of Kanamycin, shaking overnight at 37°C. The next day, the inoculum was diluted in one liter of TB and incubated at 37°C, shaking until its OD600 reached 0.8. Then, 1 mM IPTG (ThermoFisher, Cat number: R0393) was added to induce protein production, and the bacteria were returned to 37°C for another 3 hours of shaking. The bacteria were collected, and the pellet was stored at -80°C. The next day, the pellet was lysed using lysis buffer (pH: 7.4) containing 20 mM sodium phosphate, 500 mM sodium chloride, 10 mM imidazole, lysozyme (Img per 1 ml of lysis buffer), %1 triton-X andprotease inhibitor tablets (cOmplete ULTRA Tablets, Mini, EDTA-free, EASYpack Protease Inhibitor Cocktail, Sigma, Cat number: 5892791001) on ice for 30 minutes, followed by sonication (10 seconds on, 30 seconds off, total 1 minutes in %50 of power). The lysate was centrifuged at 23,000xg for 20 minutes at 4°C, and the clear lysate was loaded onto the gravity column containing 10 ml of Ni-NTA Agarose (Qiagen, Cat number: 30230). The batch-binding was done for 2 hours at 4°C, and the protein was eluted with 250 mM of Imidazole. Immediately after elution, the protein was buffer-exchanged by 50 kDa Amicon tubes, and the concentrated proteins were loaded onto AKTA pure-25 to do size-exclusion chromatography (SEC) using Superdex 75 10 / 300 GL column (Cytiva, Cat number: 17517401) and the peaks were run on the gel, stained with Coomassie Blue and pure fractions were snap-freezed and stored at -80°C.

[0316] GP38 was purified from ExpiCHO cells as follows. After thawing, ExpiCHO cells were subjected to at least three passages prior to transfection. A day before the transfection, the cells were counted and diluted to 1 million / ml by FectoCHO CD Expression Medium supplemented by 10 mM L-Glutamine. The next day, pGSCD-Mucin-gp38 plasmid and Furin (FURIN (NM_002569) Human Untagged Clone, Origene, Cat number: SCI 18550) expressing plasmids at the ratio of 4:1 was added to FectoCHO CD Expression Medium followed by adding FectoPro reagent (Sartorius, Cat number: 101000007) according to its manual and after 10 minutes incubation at room temperature, the DNA / FectoPro mixture was added to cells and the supernatant was collected when the cell viability dropped below %70. The biotin from the collected supernatant was separated by adding BioLock (IBA, Cat number: 2-0205-250), shaking at room temperature for 20 minutes, followed by centrifugation at 3250xg for 20 minutes. The supernatant was filtered through a 0.22pM filter and then Buffer W (lOx) (IBA, Cat number: 2-1003-100) was added in the ratio of 1 : 10 to adjust the PH before starting the purification. Then the supernatant was added to the gravity column containing 5 ml of Strep- TactinXT 4Flow resin (IBA, Cat number: 2-5010-025) and the protein was purified by elusion with IX Buffer BXT (IBA, Cat number: 2-1042-025) as recommended by its manual. The collected protein was subjected to SEC as described for nucleocapsid. mRNA Synthesis and Purification

[0317] To generate the mRNAs, three different cap structures were utilized: ARCA, AG, and M6. For the first animal experiment, ARCA-capped mRNAs expressing wild-type nucleocapsid with three different untranslated regions (UTRs) (CCHFV, T, and ACTA-1) wereprepared using the HyperScribe Co-transcription mRNA Synthesis Kit Plus (ARCA, 5mCTP, yUTP, T7) (ApexBio, catalog no. KI 407) according to the manufacturer’s protocol. Briefly, plasmid templates were digested for one hour at 65°C with BstBI enzyme and subsequently purified using the DNA Clean & Concentrator-25 kit (Zymo Research, catalog no. D4034). One microgram of purified DNA was used as a template in a 25 pL reaction to generate ARC A- capped mRNAs containing 5-methyl-CTP and pseudouridine-UTP modifications. Doublestranded RNA contaminants were removed using a cellulose-based purification method as previously described, and the final mRNAs were further purified using the Monarch Spin RNA Cleanup Kit (500 pg) (New England Biolabs, catalog no. T2050L) and stored at -80°C.

[0318] For the second animal experiment, AG-capped mRNAs expressing nucleocapsid, Nmut, GP85, and GP85 (GP38Aglyc) were synthesized using the mMESSAGE mMACHINE T7 mRNA Kit with CleanCap Reagent AG (Thermo Fisher Scientific, catalog no. A57620) according to the manufacturer’s instructions, except that TheraPure GMP Nl- methylpseudouridine-UTP (Thermo Fisher, catalog no. R0491SKB007) was substituted for normal UTP. For the third animal experiment, CleanCap Reagent M6 (TriLink Biotechnologies, catalog no. N-7453-10) was used to generate M6-capped mRNAs. All M6- capped mRNAs were synthesized in a 1 Ox buffer containing Tris-HCl (pH 7.5, 400 mM), HC1 (150 mM), MgCh (160 mM), DTT (100 mM), and spermidine (21.2 mM), following the manufacturer’s recommendations, except that TheraPure GMP Nl-methylpseudouridine-UTP (Thermo Fisher, catalog no. R0491SKB007), 5-methylcytidine-5'-triphosphate (TriLink, catalog no. N-1014-10), and CleanScribe RNA Polymerase (TriLink, catalog no. E-0107-08) were used. Double-stranded RNA removal and RNA purification were performed as described previously for ARCA- and AG-capped mRNAs.

[0319] The integrity of the synthesized mRNAs was verified by native agarose gel electrophoresis. Specifically, 1% low-melting agarose gels were prepared in l x TAE buffer containing SYBR Safe dye. mRNA samples (1 pg / well) were mixed with an equal volume of 2x RNA loading dye (Thermo Fisher, catalog no. SMI 823) and heated at 70°C for 5 minutes prior to loading. Gels were run for 45 minutes at 120 V and visualized using a ChemiDoc MP imaging system (Bio-Rad).In vitro Expression of mRNAs

[0320] The expression of M6 capped mRNAs was assessed by western blot in HEK293T, C2C12, and HepG2. Briefly, the day before, cells were cultured in 6-well plates in completemedia. On the day of transfection, the supernatant was removed and replaced by 1 ml of Opti- MEM one hour before transfection. The transfection was performed using TransIT-mRNA Transfection Kit (Minis bio, Cat number: MIR 2225) according to their manual and the cells and supernatant were collected after 18 hours to assess the expression of target proteins by western blot (WB). Briefly, the lysate (For nucleocapsid) and supernatant (for GP38) were mixed 1 : 1 with 2x Laemmli sample buffer and boiled at 90°C for 5 minutes before loading and running on 4-20% Mini-PROTEAN TGX Precast Protein Gels. Then the gel was transferred to the nitrocellulose membrane and blocked for 30 minutes with the blocking buffer (%5 milk in %0.1 PBS-Tween) at room temperature (RT). Primary antibodies (Anti -Nucleoprotein Crimean-Congo hemorrhagic fever virus antibody (1 :3000, Abeam, Cat number: abl90657), and Monoclonal Anti-Crimean-Congo Hemorrhagic Fever Virus Pre-Gn Glycoprotein, Clone 13G8 (produced in vitro) (1 : 2500, BEI, Cat number: NR-40294) were added to the membrane in the blocking buffer for one hour at RT while shaking. Following 3 wash steps, each 5 minutes with %0.1 PBS-Tween, the secondary antibodies (Anti -Mouse IgG (H+L), HRP Conjugate, Promega, Cat number: W4021 for nucleocapsid and Goat Anti -Rabbit IgG H&L (HRP), abeam, Cat number: ab6721 for GP38) were added for 1 hour at RT in blocking buffer, and the bands were visualized by SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher, Cat number: 34094).LNP production and characterization

[0321] Purified mRNAs were encapsulated using Flex-M microfluidics (PreciGenome). Briefly, the aqueous phase containing 478.1 pg of mRNA in 3 mL of 100 mM sodium citrate (PH 5.1) was mixed with 1 mL ethanol phase containing DSPC (BROADPHARM, Cat number: BP-25623), cholesterol (Sigma, Cat number: C8667), BP-104 (BROADPHARM, Cat number: BP-26362), and DMG-PEG2000 (BROADPHARM, Cat number: BP-25496) (10:38.5:50: 1.5) in a flow rate ratio of 3: 1 and N / P ratio of 6. The collected mRNA / LNP was immediately transferred to 50 ml of ice cold IxPBS and centrifuged in 50K Amicon tubes to buffer-exchange, concentrated and stored at -80°C. To remove potential particulates, samples were filtered using 0.2 pm PVDF Titan syringe filters (42204-PV, Thermo Fisher Scientific) attached to sterile 1 mL plastic syringes.

[0322] LNP size distribution, zeta potential, and poly dispersity (PI) were assessed using dynamic light scattering (DLS) with a Zetasizer Pro Red instrument (Malvern Panalytical, UK). For measurement, 10 pL of thoroughly washed LNP suspension was diluted in 1 mL of lxPBS. Each sample was measured in triplicate, and results are reported as the average hydrodynamic diameter ± standard deviation.

[0323] The encapsulation efficiency of mRNA within LNPs was evaluated using the Quant-iT RiboGreen RNA Assay (Thermo Fisher Scientific, Cat number: rl l490) as previously described. Fluorescence measurements were performed in black opaque 96-well plates (CulturPlate-96 F, Perkin Elmer, USA) using a SpectraMax iD3 to an excitation wavelength of 485 nm (15 nm bandpass) and an emission wavelength of 528 nm (20 nm bandpass).

[0324] The apparent pKa of LNPs was determined using the LipidLaunch LNP Apparent pKa Assay Kit (TNS Method) (Cayman Chemical, Cat number: 702680). Briefly, LNPs were diluted to a final concentration of 500 nM and incubated with 6 pM TNS in a total volume of 150 pL. The assay was performed in a series of buffered solutions spanning a pH range from 3 to 10. Each buffer solution contained 20 mM boric acid, 10 mM imidazole, 10 mM sodium acetate, 10 mM glycylglycine, and 25 mM NaCl to maintain consistent ionic strength across the pH range. Fluorescence was measured using SpectraMax iD3 with excitation and emission wavelengths set at 321 nm and 445 nm, respectively. The pH of each well was recorded following TNS addition to ensure accurate assignment of fluorescence values. The resulting fluorescence data were fitted to the Henderson-Hasselbalch equation using Mathematica (Wolfram Research) to calculate the apparent pKa of the LNP formulation.Antigen Presentation by Primary Dendritic Cells to Activate T cells in vitro

[0325] An in vitro co-culture experiment was designed to evaluate the antigen presentation potential of dendritic cells to T cells and to compare lipid nanoparticle (LNP) formulations containing the ionizable lipid BP- 104 with those prepared using FDA-approved ionizable lipids SM-102 (BroadPharm, catalog no. BP-25499) and ALC-0315 (BroadPharm, catalog no. BP- 25498). Briefly, CD1 lc+MHCIE dendritic cells were isolated from the spleens of naive 8-10- week-old female C57BL / 6 mice using the EasySep Mouse CDl lc Positive Selection Kit II (STEMCELL Technologies, catalog no. 18781) according to the manufacturer’s instructions. The isolated cells were resuspended in RPMI medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, 0.1 mM P-mercaptoethanol, and 2 mM L-glutamine. Dendritic cells were plated in untreated 6-well plates at a density of 5 x io5cells per well in 1 mL of complete medium. Each well received 5 pg of mRNA / LNPs containing Nmut mRNA encapsulated in LNPs formulated with BP-104, SM-102, or ALC-0315. A positive controlconsisting of 100 ng / mL lipopolysaccharide (LPS) from Escherichia coli (O55:B5) (STEMCELL Technologies, catalog no. 100-1270) was included.

[0326] After a 24-hour incubation, the cells were harvested and washed twice with 1 x PBS to remove extracellular antigens. A total of 1.5 x io4dendritic cells were transferred to each well of an untreated 96-well plate. CD4+(CD4+CD44AlowCD62LAhigh) T cells were isolated from naive 8-10-week-old female C57BL / 6 mouse spleens using the Easy Sep Mouse Naive CD4+T Cell Isolation Kit (STEMCELL Technologies, catalog no. 19765). The isolated T cells (1.5 x io5per well) were added to wells containing the stimulated dendritic cells at a 1 : 1 ratio in a total volume of 200 pL. The co-cultures were incubated for 4 days at 37°C, followed by the addition of Brefeldin A solution (BioLegend, catalog no. 420601) at a 1 :1000 dilution, and incubation for an additional 5 hours at 37°C. Cells were then collected and stained for intracellular cytokines as described in the section on flow cytometric analysis of T cells.

[0327] Supernatants were collected for cytokine and chemokine quantification using the LEGENDplex Mouse Anti-Virus Response Panel (13-plex) kit (BioLegend, catalog no. 740622). The assay was performed following the manufacturer’s instructions using the provided V-bottom 96-well plate. Briefly, assay buffer, standards, and supernatants were added to the wells, followed by fluorescently encoded capture beads conjugated with analyte-specific antibodies. After a 2-hour incubation at room temperature on a plate shaker protected from light, the plate was washed to remove unbound material. A biotinylated detection antibody cocktail was then added to form antibody-antigen-antibody sandwich complexes. After a 1- hour incubation, streptavidin-PE was added to bind the biotinylated detection antibodies, generating a fluorescent signal proportional to the analyte concentration. Following two wash steps, the beads were resuspended in assay buffer for acquisition. Samples were analyzed on a flow cytometer equipped with 488 nm and 633 nm lasers and appropriate filter settings for detection of both bead regions (forward / side scatter and APC channel) and PE signal. A minimum of 300 events per bead population were acquired, and data were analyzed using BioLegend LEGENDplex Data Analysis Software.In Vivo Immunogenicity Experiment

[0328] All animal procedures were conducted in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the Broad Institute of MIT and Harvard (protocol number 0376-09-23). Mice were housed under specific pathogen-free (SPF) conditions in ventilated, ducted Innovive racks and maintained on a 12-hour light / dark cycle. Environmental parameters, including temperature (70-72°F) and relative humidity (30-40%), were regulated according to The Guide for the Care and Use of Laboratory Animals (National Research Council). Female C57BL / 6 and BALB / c mice (Jackson Laboratory, Bar Harbor, ME), aged 8-12 weeks, were randomized into experimental groups and immunized intramuscularly with 10 pg of mRNA-lipid nanoparticle (LNP) vaccine formulations in 50 pL of sterile l x PBS. BALB / c mice were used exclusively to evaluate antibody responses for the ARCA-capped mRNA constructs.

[0329] In all immunogenicity experiments, a booster immunization regimen was employed, consisting of an initial dose on Day 0 followed by a booster on Day 14. Mice were euthanized on Day 28 for ARCA- and AG-capped constructs and on Day 32 for Cleancap M6 constructs. As negative controls, mice were injected with l x PBS for ARCA and AG groups, and with iLNP (empty LNP) for Cleancap M6 groups.

[0330] For ARCA-capped mRNA / LNPs, three different mRNAs encoding nucleocapsid with distinct untranslated regions (UTRs) — CCHFV, T, and ACTA-1 — were administered to C57BL / 6 mice. Nucleocapsid (N) and mutant nucleocapsid (Nmut) ARCA-capped mRNA / LNPs were administered to BALB / c mice to evaluate antibody responses. Vaccine constructs for AG and Cleancap M6 experiments in C57BL / 6 mice encoded one of the following: full-length nucleocapsid, mutant nucleocapsid (Nmut), GP85, or GP85 containing deglycosylated GP38 (GP38Aglyc). Each vaccine dose consisted of 10 pg of mRNA-LNP diluted in sterile l x PBS to a final volume of 50 pL and administered intramuscularly into the left thigh on Day 0, followed by a booster injection on Day 14.

[0331] Blood samples were collected 2-3 weeks after the booster dose via terminal cardiac puncture under deep anesthesia. Serum was isolated by centrifugation at 1,000 x rpm for 15 minutes at 4°C, aliquoted, and stored at -80°C until further analysis.In-House Enzyme-Linked Immunosorbent Assay

[0332] Binding antibodies against nucleocapsid and GP38 were measured by designing an in-house ELISA assay. Antigens (nucleocapsid or GP38) were coated overnight at 4°C in Immulon 2 HB at a concentration of 5pg / ml of lx PBS (lOOpl / well). The next day, the antigens were removed, and the plates were washed one time with %0.1 Tween-PBS (300pl / well) and blocked by 200 pl of ChonBlock ELISA Buffer (Chondrex, Cat number: 9068) at room temperature for 2 hours. Following 3 washes with the wash buffer, the two times diluted serum samples in the blocking buffer (lOOpl / well) were added to each well and incubated at roomtemperature for 2 hours. After 4 washes, the secondary antibodies (Anti-Mouse IgG (H+L), HRP Conjugate, Promega, Cat number: W4021) were 1 :3000 diluted in ChonBlock Detection Antibody Dilution ELISA Buffer (Chondrex, Cat number: 90681) and added to each well (lOOpl / well) and incubated at room temperature for one hour. The plate was washed four times with the wash buffer, and lOOpl of 1-Step TMB ELISA Substrate Solution was added to each well. After 5-15 minutes, the reaction was stopped by adding 100 pl of stop solution, and the plates were read at 450 nm in SpectraMax iD3. For result analysis, the end-point titer was determined as the highest serum dilution producing an optical density (OD) value exceeding the mean plus two standard deviations of the negative control wells, which was considered positive.Neutralization Assay by Pseudotyped Vesicular Stomatitis Virus Expressing Modified Glycoprotein of CCHFV

[0333] Replication-incompetent pseudotyped vesicular stomatitis virus (VSV) expressing a modified full-length M segment of CCHFV was generated to assess the presence of neutralizing antibodies in mice immunized with GP38 and GP38Aglyc constructs. The sequence of this modified M segment is described elsewhere. The gene encoding the modified M segment was synthesized by GenScript and cloned into a pcDNA3.1(+) plasmid to generate pGSCD-VSV-M.

[0334] To produce the pseudotyped VSV-M particles, HEK293T cells were seeded into 6- well plates one day prior to transfection. On the following day, the medium was replaced with 1 mL per well of DMEM, and the transfection reaction was prepared following the manufacturer’s protocol using pGSCD-VSV-M and pCAGGS-G-Kan plasmids (Kerafast, catalog no. EH1017). The transfection mixture was added to the cells, and after 6 hours, the medium was replaced with DMEM supplemented with 10% fetal bovine serum (FBS). After 24 hours, pseudotyped AG-luciferase (G*AG-luciferase) recombinant VSV (Kerafast, catalog no. EH1020-PM) was added to each well and incubated for 1 hour at 37°C. The wells were subsequently washed with l x PBS and overlaid with 2 mL DMEM containing 2% FBS. For VSV-M production, an anti-G VSV antibody (Kerafast, catalog no. EB0010) was included at a 1 : 1000 dilution to neutralize residual rVSV-G.

[0335] The supernatant containing pseudotyped virus was collected 24 hours postinfection, aliquoted, and stored at -80°C. For the neutralization assay, serum samples were serially twofold diluted starting at a 1 : 100 dilution. Fifty microliters of diluted serum weremixed with 50 pL of pseudotyped virus and incubated for 1 hour at 37°C. The virus-serum mixtures were then transferred to 96-well plates containing Vero cells. After 1 hour of incubation, the inoculum was removed, and the wells were washed with l x PBS and overlaid with 100 pL phenol red-free DMEM containing 2% FBS. Following 24 hours of incubation, 100 pL of Bright-Glo reagent (Promega, catalog no. E2620) was added to each well, and luminescence was measured using a SpectraMax iD3 plate reader.Enzyme-Linked ImmunoSpot (ELISpot) Assay

[0336] To evaluate antigen-specific T cell responses, IFN-y and IL-2 (for ARC A cap experiment) and IFN-y and IL-4 (for AG and Clean cap M6 experiments) ELISPOT assays were performed using splenocytes collected from immunized mice 2-3 weeks after the booster immunization. Splenocytes (from vaccine and negative groups) were isolated under sterile conditions and resuspended in a complete RPMI medium containing %10 FBS, 1% penicillinstreptomycin and 0.1 mM betamercaptoethanol and were plated in 96-well polyvinylidene fluoride (PVDF)-backed ELISPOT plates (XEL485, R&D Systems) pre-coated with an antimouse IFN-y or IL-2, or IL-4 capture antibody (R&D Systems). Cells were stimulated with lOp / ml of SEC purified nucleocapsid and GP38 proteins in the respective groups for 24 hours at 37 °C with 5% CO2 to allow cytokine secretion. After incubation, cells were gently removed, and wells were washed with ELISpot wash buffer (R&D Systems, Cat number: 895308,) to eliminate residual cells and debris. A biotinylated anti-mouse IFN-y, or -IL-2, or -IL-4 detection antibody (R&D Systems) was added and incubated for 2 hours at room temperature on a rocking platform. Subsequently, plates were incubated with streptavidin-alkaline phosphatase conjugate (R&D Systems, Cat number: 895358) for another 2 hours, followed by development with BCIP / NBT substrate solution (R&D Systems, Cat number: 895867) for 1 hour. Plates were rinsed thoroughly with distilled water to stop the reaction and allowed them to dry completely at 37°C for 30 minutes. Spot-forming units (SFUs) representing cytokinesecreting cells were enumerated using an automated ImmunoSpot analyzer (Cellular Technology Ltd). Results were expressed as SFUs per seeded cell numbers after background subtraction from unstimulated wells.Flow cytometry analysis of T, B, and Dendritic cellsT Cells

[0337] Spleens were harvested and homogenized into single-cell suspensions by passing them through a 70 pm cell strainer in the complete RPMI 1640 medium containing %10 FBS,1% penicillin-streptomycin and 0.1 mM betamercaptoethanol. The cell suspensions were centrifuged, and red blood cells were lysed using ACK lysis buffer to yield clear single-cell preparations.

[0338] For antigen-specific stimulation, 2 * 106splenocytes were cultured in 96-well plates in the presence of 5 pg mF1nucleocapsid or GP38 at 37 °C, 5% CO2. Brefeldin A (5 pg ml"1, BioLegend, Cat number: 420601) was immediately added, and incubation continued for 6 hours. DMSO-treated cells served as negative controls, while cells stimulated with 50 ng ml1phorbol 12-myristate 13-acetate (PMA) and 1 pg ml"1ionomycin were used as positive controls. After a total of 6 h, cells were washed with lx PBS, stained with Live / Dead eFlour 506 Fix Viability dye (ThermoFisher, Cat number: 65-0866-14) for 30 min at 4°C, and surface staining was then performed by antibody cocktails containing BD Pharmingen™ Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block™) (BD, Cat number: 553142), CD3-PE-Cy5 (BioLegend, Cat number: 100274), CD4-PE (ThermoFisher, Cat number: 12-0043-83), CD8a- FITC (BD, Cat number: 553031), CD45R-B220-SuperBright 600 (ThermoFisher, Cat number: 63-0452-82) for 30 min at 4°C in dark. Following surface staining, cells were washed in FACS buffer, fixed, and permeabilized using the Cytofix / Cytoperm kit (BD Biosciences, #554714) for 20 minutes at 4°C, and then stained intracellularly for 20 min using IFN-gamma-PE-Cy7 (BioLegend, Cat number: 163508), IL-2-APC-Cy7 (BD, Cat number: 560547), IL-4-PE- CF594 (BD, Cat number: 562450), TNF- Al exaFlour 700 (BioLegend, Cat number: 506338) and IL-17A Monoclonal Antibody (eBiol7B7), PerCP-Cyanine5.5(eBioscience, Cat number: 45-7177-82). After final washes, cells were analyzed on a CytoFlex-M flow cytometer equipped with four lasers.B, TfH and Dendritic Cells

[0339] Mouse inguinal lymph nodes were collected and processed into single-cell suspensions by mechanical dissociation through a 40 pm cell strainer in the complete high- glucose DMEM medium. The cell suspensions were centrifuged at 300 * g for 5 min and then washed twice with lx PBS and counted.

[0340] For GC B and TfH cells staining, 2 x io6cells per sample were stained with FVS575V fixable viable dye (BD, Cat number: 565694) for 20 minutes at 4°C. Following washing with FACS buffer (1% FBS in lx PBS), the cells stained with antibody cocktails containing BD Pharmingen™ Purified Rat Anti -Mouse CD16 / CD32 (Mouse BD Fc Block™) (BD, Cat number: 553142), CD19-NovaFlour Red 710 (ThermoFisher, Cat number:M004T02R04-A), CD3-APC-eFlour 780 (ThermoFisher, Cat number: 47-0031-82), CD4- NovaFlourYellow 690 (ThermoFisher, Cat number: M001T02Y05-A), CD95-AlexaFlour 647 (BD, Cat number: 563647), GL7-PE (BioLegend, Cat number: 144608), CD185-PE-Vio615 (Miltenyi Biotec, Cat number: 130-107-656), CD184 (CXCR4)-(2B11)-PE-Cyanine7 (ThermoFisher, Cat number: 25-9991-82), CD279 (PD-l)-(J43)-PerCP-eFluor™ 710 (ThermoFisher, Cat number: 46-9985-82), CD86-SuperBright 645 (ThermoFisher, Cat number: 64-0862-82), CCR6-Brilliant Violet 510 (BD, Cat number: 747832), nucleocapsid / GP38-AF405 and -AF488 for 30 minutes at 4 °C. To generate the nucleocapsid- or GP38- AF405 and -AF488, Alexa Fluor 488 NHS Ester (Succinimidyl Ester) (ThermoFisher, Cat number: A20000) and Alexa Fluor 405 NHS Ester (Succinimidyl Ester) (ThermoFisher, Cat number: A30100) were used according to the manufacturer manual and purified using Zeba Spin Desalting Columns, 7K MWCO (ThermoFisher, Cat number: 89877) and stored at 4°C for up to one month. After staining, cells were washed twice with FACS buffer and fixed in 300 pl of 1% paraformaldehyde. Data acquisition was performed using a CytoFlex-M flow cytometer equipped with four lasers.

[0341] For dendritic cell staining, cells were incubated with Fixable Live / Dead Ghost Dye Violet 540 (Cell Signaling Technology, catalog no. 72086) for 30 minutes at 4°C in the dark. After washing with fluorescence-activated cell sorting (FACS) buffer, the cells were incubated with the following antibodies and reagents: BD Pharmingen Purified Rat Anti-Mouse CD16 / CD32 (Mouse BD Fc Block) (BD Biosciences, catalog no. 553142), CD3-APC-eFluor 780 (Thermo Fisher Scientific, catalog no. 47-0032-82), CD19-APC-eFluor 780 (Thermo Fisher Scientific, catalog no. 47-0193-82), NKl.l-APC-eFluor 780 (Thermo Fisher Scientific, catalog no. 47-5941-82), Ly-6G-APC-eFluor 780 (Thermo Fisher Scientific, catalog no. 47- 9668-82), CDl lc-Vio Bright B515 (Miltenyi Biotec, catalog no. 130-129-314), MHCII-PE Vio615 (Miltenyi Biotec, catalog no. 130-112-393), Siglec-H-APC (REAfinity, Miltenyi Biotec, catalog no. 130-112-297), CD317-PE (BioLegend, catalog no. 127104), and XCR1- Vio Bright R720 (Miltenyi Biotec, catalog no. 130-132-757).

[0342] The staining reaction was carried out for 30 minutes at 4°C in the dark. Cells were subsequently fixed with 1% paraformaldehyde for 30 minutes at 4°C and analyzed using a CytoFLEX-M flow cytometer (Beckman Coulter).Data Analysis

[0343] All data was organized and processed using Microsoft Excel 2023. Statistical analyses were performed using GraphPad Prism version 10.0.0 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Paired comparisons (e.g., longitudinal measurements within the same animal group) were analyzed using the two-tailed Wilcoxon signed-rank test. For other datasets requiring evaluation of mean differences among multiple groups, one-way ANOVA followed by Tukey's post hoc multiple comparison tests were applied. All the antibody panels for flow cytometry experiments were designed using FluoroFinder online tool (fluorofmder.com). All the plasmids, DNA fragments, and primers used in this study were designed in silico using SnapGene software (snapgene.com). QIAGEN CLC Main Workbench 21.0 (QIAGEN, Aarhus, Denmark) was used to analyze and generate the secondary structures of the untranslated regions (UTRs) of the mRNAs. Results are reported as mean ± standard deviation (SD), and statistical significance was defined as / ? < 0.05.***

[0344] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention, as claimed, should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

Claims

CLAIMSWhat is claimed is:

1. A lipid particle comprising a compound of Formula Ior a pharmaceutically acceptable salt thereof, whereinA1and A2are each independently unsubstituted C1-C12 alkylene.

2. The lipid particle of claim 1, wherein A1and A2are each independently unsubstituted C3-, Ce-, or C9-alkylene.

3. The lipid particle of claim 1 or 2, wherein A1is unsubstituted Ce-alkylene.

4. The lipid particle of any one of claims 1-3, wherein unsubstituted A2is Ce-alkylene.

5. The lipid particle of claim 1 or 2, wherein A1is unsubstituted C9-alkylene.

6. The lipid particle of claim 1, 2, or 5, wherein A2is unsubstituted Cg-alkylene.

7. A lipid particle comprising heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2- hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof.

8. The lipid particle of any one of claims 1-7 further comprising an oligonucleotide.

9. The lipid particle of claim 8, wherein the oligonucleotide is antisense and / or messenger RNA (mRNA).

10. The lipid particle of claim 9, wherein the mRNA is a chemically modified mRNA sequence, optionally comprising N-methyl-pseudouridine triphosphate (N-methylpseudo- UTP) and / or 5-methyl-cytidine triphosphate (5-methyl-CTP).

11. The lipid particle of claim 9 or 10, wherein the mRNA comprises a coding region that encodes at least one antigenic polypeptide or protein comprising a sequence modification, optionally selected from nucleocapsid, GP38, or a mutant thereof.

12. The lipid particle of any one of claims 9-11, wherein the mRNA additionally comprises a) a 5’-UTR structure, b) a poly(A) sequence, c) a poly (C) sequence, d) a 3’-UTR structure, e) a 5’ cap; or a combination thereof.

13. The lipid particle of claim 12, wherein a 5'-UTR structure is selected from CCHFV- UTR, T-UTR, and ACTA-UTR.

14. The lipid particle of claim 12 or 13, wherein the 3’-UTR structure is selected from CCHFV-UTR, T-UTR, and ACTA1-UTR.

15. A pharmaceutical composition comprising the lipid particle of any one of claims 1-14 and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof.

16. A pharmaceutical composition comprising a compound of Formula IFormula I or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof, whereinA1and A2are each independently unsubstituted CI-CH alkylene.

17. The pharmaceutical composition of claim 16, wherein A1and A2are each independently unsubstituted C3-, Ce-, or C9-alkylene.

18. The pharmaceutical composition of claim 16 or 17, wherein A1is unsubstituted Ce- alkylene.

19. The pharmaceutical composition of any one of claims 16-18, wherein unsubstituted A2is Ce-alkylene.

20. The pharmaceutical composition of claim 16 or 17, wherein A1is unsubstituted C9- alkylene.

21. The pharmaceutical composition of claims 16, 17, or 20, wherein A2is unsubstituted C9-alkylene.

22. A pharmaceutical composition comprising heptadecan-9-yl 7-((7-(heptyloxy)-7- oxoheptyl)(2-hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof.

23. The pharmaceutical composition of any one of claims 15-22, comprising one or more neutral lipids, steroids, or polymer-conjugated lipids.

24. The pharmaceutical composition of claim 23, wherein the one or more neutral lipids are selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

25. The pharmaceutical composition of claim 23 or 24, wherein the neutral lipid is DSPC.

26. The pharmaceutical composition of any one of claims 23-25, wherein a molar ratio of compound to neutral lipid is about 2: 1 to about 8: 1.

27. The pharmaceutical composition of any one of claims 23-26, wherein the steroid is cholesterol.

28. The pharmaceutical composition of any one of claims 23-27, wherein the molar ratio of compound to steroid is about 5 : 1 to about 1 :1.

29. The pharmaceutical composition of any one of claims 23-28, wherein the one or more polymer-conjugated lipids comprise one or more pegylated lipids.

30. The pharmaceutical composition of claim 29, wherein the molar ratio of compound to pegylated lipid is about 100: 1 to about 20: 1.

31. The pharmaceutical composition of claim 29 or 30, wherein the one or more pegylated lipids are selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, PEG dialkyoxypropylcarbamate, or a combination thereof.

32. The pharmaceutical composition of claim 29 or 30, wherein the one or more pegylated lipids is a compound of Formula (II)whereinR1and R2are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds and n has a mean value of about 30 to about 60.

33. The pharmaceutical composition of claim 32, wherein R1and R2are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.

34. The pharmaceutical composition of claim 32 or 33, wherein n has a mean value of about 45.

35. The pharmaceutical composition of any one of claims 32-34, wherein the one or more pegylated lipids further comprises 1-monom ethoxypoly ethylenegly col-2, 3- dimyri stylgly cerol .

36. A method for prevention or treatment of cancer or tumor diseases, infectious diseases, allergies, or autoimmune diseases or disorders related thereto, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to a subject.

37. The method of claim 36, wherein the infectious disease is a viral, bacterial, parasitic, or fungal infection.

38. The method of claim 37, wherein the viral infection is selected from adenovirus, Crimean-Congo hemorrhagic fever, hepatitis A, hepatitis B, human papillomavirus (HPV), seasonal flu, Japanese encephalitis, measles, mumps, polio, rabies, respiratory syncytial virus, rotavirus, rubella, SARS-CoV-2, shingles, smallpox, typhoid fever, varicella, and yellow fever.

39. The method of claim 37, wherein the bacterial infection is selected from anthrax, cholera, diphtheria, Haemophilus influenzae type b (Hib), meningitis, pertussis, pneumonia, tetanus, tuberculosis, and typhoid fever.

40. A method of administering a therapeutic agent to a subject, comprising providing a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

41. A method for expressing a protein in a subject, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

42. The method of claim 41, wherein the protein is encoded by an mRNA expressed in the subject.

43. A method for vaccinating a subject against a viral pathogen, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

44. The method of claim 43, wherein the viral pathogen is selected from an Adenoviridae , a Coronavirus, an enterovirus, a flaviviridae, a hepadnaviridae , a hepeviridae, an incertae sedis, a lyssavirus, a morbillivirus, an orthomyxoviridae, an orthonairovirus, an orthopneumovirus, an orthopoxvirus, an orthorubulavirus, a papillomaviridae, a picornaviridae, rotavirus, a rubivirus, and a varicellovirus .

45. The method of claim 43, wherein the viral pathogen is selected from Adenovirus, Crimean-Congo hemorrhagic fever virus, Hepacvirus, Hepatitis B virus, Hepatitis delta virus, Hepatovirus A, Human alphaherpesvirus 3, Human papillomavirus, H1N1, H3N2, Influenza A virus, Influenza B virus, Influenza C virus, Influenza D virus, Japanese encephalitis virus, Measles morbillivirus, Mumps orthorubulavirus, Orthohepevirus A, Poliovirus, Rabies lyssavirus, Rotavirus A, Rotavirus B, Rotavirus C, Rotavirus D, Rotavirus F, Rotavirus G, Rotavirus H, Rotavirus I, Rotavirus J, Rubivirus rubellae, Severe acute respiratory syndrome-related coronavirus, Variola virus, and Yellow fever virus.

46. The method of claim 43, wherein the viral pathogen is Crimean-Congo hemorrhagic fever virus.

47. The method of claim 43, wherein the viral pathogen is Severe acute respiratory syndrome-related coronavirus.

48. The method of claim 43, wherein the viral pathogen is H1N1, H3N2, Influenza A virus, Influenza B virus, or a combination thereof.

49. A method for vaccinating a subject against a bacterial pathogen, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

50. The method of claim 49, wherein the bacterial pathogen is selected from a Bacillus, a Bordetella, a Clostridium, a Corynebacterium, Haemophilus, a Mycobacterium, a Neisseria, a Salmonella, a Streptococcus, and a Vibrio.

51. The method of claim 49 or 50, wherein the bacterial pathogen is selected from B. anthracis, B. pertussis, C. diphtheriae, C. tetani, H. influenzae, M. tuberculosis, N. meningitidis, S. enterica, S. pneumoniae, and V. cholerae.

52. A method for vaccinating a subject against Crimean-Congo hemorrhagic fever, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

53. A method for vaccinating a subject against SARS-CoV-2, comprising administering a lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to the subject.

54. A method for vaccinating against seasonal flu, comprising administering the lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to a subject.

55. A method of preventing or treating cancer, comprising administering the lipid particle according to any one of claims 1-14 or a pharmaceutical composition according to any one of claims 15-34 to a subject, wherein the lipid particle or pharmaceutical composition induces an immunogenic response in the subject56. The method of claim 55, wherein the cancer is selected from adenocarcinoma, bladder cancer, breast cancer, carcinoma, colon cancer, colorectal cancer, gastroesophageal cancer, glioblastoma, head and neck cancer, hepatocellular cancer, lung cancer, lymphoma, ovarian cancer, pancreatic cancer, rectal cancer, skin cancer, solid tumors, and urothelial cancer.

57. The method of any one of claims 1—56, wherein the subject is human.

58. A vaccine composition comprising heptadecan-9-yl 7-((7-(heptyloxy)-7-oxoheptyl)(2- hydroxyethyl)amino)heptanoateor a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, excipients, or a combination thereof.

59. The vaccine composition of claim 58, comprising one or more neutral lipids, steroids, or polymer-conjugated lipids.

60. The vaccine composition of claim 59, wherein the one or more neutral lipids are selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

61. The vaccine composition of claim 59 or 60, wherein the neutral lipid is DSPC.

62. The vaccine composition of any one of claims 59-61, wherein a molar ratio of compound to neutral lipid is about 2: 1 to about 8: 1.

63. The vaccine composition of any one of claims 59-62, wherein the steroid is cholesterol.

64. The vaccine composition of any one of claims 59-63, wherein the molar ratio of compound to steroid is about 5 : 1 to about 1 : 1.

65. The vaccine composition of any one of claims 59-64, wherein the one or more polymer-conjugated lipids comprise one or more pegylated lipids.

66. The vaccine composition of claim 65, wherein the molar ratio of compound to pegylated lipid is about 100: 1 to about 20: 1.

67. The vaccine composition of claim 65 or 66, wherein the one or more pegylated lipids are selected from PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, PEG dialkyoxypropylcarbamate, or a combination thereof.

68. The vaccine composition of claim 65 or 66, wherein the one or more pegylated lipids is a compound of Formula (II)whereinR1and R2are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds and n has a mean value of about 30 to about 60.

69. The vaccine composition of claim 68, wherein R1and R2are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.

70. The vaccine composition of claim 68 or 69, wherein n has a mean value of about 45.

71. The vaccine composition of any one of claims 67-70, wherein the one or more pegylated lipids further comprises l-monomethoxypolyethyleneglycol-2,3-dimyristylglycerol.

72. The vaccine composition of any one of claims 58-71, further comprising an oligonucleotide.

73. The vaccine composition of claim 72, wherein the oligonucleotide is antisense and / or messenger RNA (mRNA).

74. The vaccine composition of claim 73, wherein the mRNA is a chemically modified mRNA sequence.

75. The vaccine composition of claim 73 or 74, wherein the mRNA comprises a coding region that encodes at least one antigenic polypeptide or protein comprising a sequence modification.

76. The vaccine composition of any one of claims 73-75, wherein the mRNA additionally comprises a) a 5’-UTR structure, b) a poly(A) sequence, c) a poly (C) sequence, d) a 3’-UTR structure, e) a 5’ cap structure, or a combination thereof77. The vaccine composition of claim 76, wherein a 5'-UTR structure is selected from CCHFV-UTR, T-UTR, and ACTA-UTR.

78. The vaccine composition of claim 76 or 77, wherein the 3’-UTR structure is selected from CCHFV-UTR, T-UTR, and ACTA1-UTR.

79. The vaccine composition of claim 73, wherein the mRNA encodes a mucin-modified gp38 mutant.

80. The vaccine composition of claim 79, wherein mucin-modified gp38 mutant is chemically modified to remove N-glycosylation sites.

81. The vaccine composition of claim 71, wherein the mRNA encodes CCHFV nucleocapsid protein wherein the DEVD motif at amino acids 266-269 is replaced with AEVA.

82. The vaccine composition of any one of claims 58-80, wherein the composition is a lipid particle composition.

83. The lipid particle of any one of claims 9-12, wherein the mRNA further comprises a 5' cap structure selected from Anti-Reverse Cap Analog (ARCA), adenosine-guanosine (AG) cap, or a base-modified Cap 1 structure.

84. The vaccine composition of any one of claims 73-83, wherein the mRNA further comprises a 5' cap structure selected from Anti -Reverse Cap Analog (ARCA), adenosineguanosine (AG) cap, or a base-modified Cap 1 structure.

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