Compositions and methods for the treatment of cancer

Abstract

The present disclosure relates to compositions and methods for the preparation, manufacture and therapeutic use of nucleic acid vaccines comprising polynucleotide sequences encoding one or more proteins, polypeptides or peptides fragments or variants thereof of tumor-associated antigens for the prevention, alleviation and / or treatment of various and other diseases, disorders and / or conditions caused by tumor-associated antigens.

Description

COMPOSITIONS AND METHODS FOR THE TREATMENT OF CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Application No.: 63 / 743,395 filed January 9, 2025 entitled Compositions and Methods for the Treatment of Cancer; US Provisional Application No.: 63 / 743,887 entitled Compositions and Methods for the Treatment of Cancer filed January 10, 2025; US Provisional Application No.: 63 / 825,094 filed June 17, 2025 entitled Compositions and Methods for the Treatment of Cancer; US Provisional Application No.: 63 / 825,114 filed June 17, 2025 entitled Compositions and Methods for the Treatment of Cancer; the contents of each of which are incorporated by reference in their entirety.SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file, entitled 1038PCTSEQLST.xml, was created on January 8, 2026, and is 388,026 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure relates to compositions, formulations, methods, and / or uses of nucleic acid vaccines, specifically nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, polypeptides or peptides fragments or variants thereof of one or more tumor-associated antigens for the prevention, alleviation and / or treatment of various and other diseases, disorders and / or conditions.

[0004] The present disclosure also relates to formulations with lipid and lipids nanoparticles (LNPs) for the delivery of nucleic acids, specifically nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, polypeptides or peptides fragments or variants thereof of one or more tumor-associated antigens and methods of use of these LNPs for the treatment of diseases, disorders and / or conditions.BACKGROUND

[0005] Although there have been significant advances in understanding various cancers caused or associated with tumor-associated antigens, the disease remains difficult to treat as standard therapies have not always been effective. There are emerging therapies that have provided some hope, but there is still a need for an effective treatment. Therefore, provided herein are nucleic acid vaccines encoding one or more proteins, polypeptides or peptides fragments or variants thereof of tumor-associated antigens for the treatment of cancers, and associated diseases, disorders and / or conditions.

[0006] mRNA-based vaccines represent a promising approach to cancer immunotherapy, leveraging the immune system to recognize and eliminate tumor cells expressing specific tumor-associated antigens. Several therapeutic mRNA vaccines in development have shown promise by eliciting T-cell responses targeting tumor-associated antigens. These vaccines offer advantages over conventional therapies by stimulating both CD8+ cytotoxic T lymphocytes (CTLs) and CD4+ helper T cells, which play a crucial role in generating a sustained anti-tumor immune response

[0007] Additionally, single antigen constructs have been the standard for therapeutics but generally they have to be used in combination with other therapeutics in order to achieve the desired therapeutic effect in cancer cells. However, vaccines encoding multiple tumor-associated antigens may support a potential synergistic effect of expression of different tumor antigens across a variety of tumor cells as compared to vaccines encoding single tumor-associated antigens.SUMMARY

[0008] The present disclosure provides nucleic acid vaccines encoding one or more tumor-associated antigens, such as MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX2, TTK, SPA17, or epidermal growth factor receptor variant III (EGFRvIII). Also provided are particles, such as nanoparticles and nanoparticles comprising the nucleic acid vaccines.

[0009] In one embodiment, provided are polynucleotides comprising a sequence encoding at least one region from each of five tumor-associated antigens, or a fragment, peptide or variant thereof, wherein said tumor-associated antigens are selected from the group consisting of MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX2, TTK and SPA17. The polynucleotide may have one or many sequence regions each comprising a sequence that is at least 80% identical to SEQ ID NO. 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, and / or 33. Further the polynucleotide may further comprise a sequence region that is at least 80% identical to SEQ ID NO. 103-114 or 128-139, a sequence region that is at least 80% identical to SEQ ID NO. 142, 145, or 148, and / or a sequence region that is at least 80% identical to SEQ ID NO. 89.

[0010] According to another aspect, the polynucleotide may comprise a nucleic acid sequence having 80% identity to SEQ ID NO. 34, 35, 38, 39, 46, 47, 50, or 51. In some aspects, the polynucleotide may comprise the nucleic acid sequence SEQ ID NO. 34, 35, 38, 39, 46, 47, 50, or 51.

[0011] According to another aspect, the polynucleotide may comprise a nucleic acid sequence having 80% identity to 42 or 43. In some aspects, the polynucleotide may comprise the nucleic acid sequence SEQ ID NO. 42 or 43.

[0012] In some embodiments, provided are polynucleotides comprising a sequence encoding epidermal growth factor receptor variant III (EGFRvIII), or a fragment, peptide or variant thereof, wherein the polynucleotide comprises a first sequence region having a nucleic acid sequence that is atleast 80% identical to SEQ ID NO. 166, 167, 168, 169 or 170. In one aspect, the first sequence region may consist of SEQ ID NO. 166. In another aspect, the first sequence region may consist of SEQ ID NO.167. In another aspect, the first sequence region may consist of SEQ ID NO. 168. In another aspect, the first sequence region may consist of SEQ ID NO. 169. In another aspect, the first sequence region may consist of SEQ ID NO. 170.

[0013] In some embodiments, the polynucleotides of the present disclosure are at least 50% codon optimized.

[0014] In some embodiments, the polynucleotides of the present disclosure are an mRNA. According to one aspect, the polynucleotide comprises a 5’UTR and a 3’UTR, wherein said 5’UTR comprises SEQ ID NO. 150 and said 3’UTR comprises SEQ ID NO. 152, 154, 156 or 158.

[0015] In some embodiments, the polynucleotides of the present disclosure comprise a nucleic acid sequence selected from the group consisting of SEQ ID NO. 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 171, 172, 174, 175, 177, 178, 180, 181, 183, and 184.

[0016] In some embodiments, the polynucleotide may comprise a nucleic acid sequence where at least one uracil nucleoside or all of the uracil nucleosides are modified to be N1 -methylpseudouridine.

[0017] According to another aspect, the nucleic acid vaccine comprises any of the polynucleotides described herein. Further, the nucleic acid vaccine may be formulated in a lipid nanoparticle (LNP). The LNP may comprise (a) from about 40 to about 100 mol% of an ionizable lipid, (b) from about 0 to about 10 mol% of a neutral lipid, (c) from about 0 to about 50 mol% of a helper lipid, (d) from about from 0 to about 5 mol% of a polymer-conjugated lipid, and (e) from 0 to about 5 mol% of a hydrophobic component, wherein the mol% are based on the total lipids present in the nanoparticle. In another embodiment, the LNP may comprise (a) from about 40 to about 60 mol% of an ionizable lipid, (b) from about 5 to about 10 mol% of a neutral lipid, (c) from about 30 to about 45 mol% of a helper lipid, (d) from about from 1 to about 54 mol% of a polymer-conjugated lipid, and (e) from 0.1 to about 5 mol% of a hydrophobic component, wherein the mol% are based on the total lipids present in the nanoparticle. In some embodiments, the ionizable lipid is Compound 201, and further the neutral lipid is DSPC, the helper lipid is cholesterol, the polymer-conjugated lipid is PEG2k-DMG and the hydrophobic component is squalene.

[0018] According to another aspect, provided herein is a pharmaceutical composition comprising a nucleic acid vaccine and a pharmaceutically acceptable excipient.

[0019] According to another aspect, provided herein are methods of inducing an immune response in a subject by administering the nucleic acid vaccine or the pharmaceutical composition described herein. The immune response may be a T-cell response.

[0020] According to another aspect, provided herein are methods of treating cancer in a subject by administering the nucleic acid vaccine or the pharmaceutical composition described herein to induce an immune response against cancer cells. In some embodiments the cancer is a solid tumor, basal cell carcinomas, bladder cancer, brain cancer, cholangiocarcinoma, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, female reproductive system-associated carcinomas, endometrial carcinomas, uterine serous carcinomas, gastric cancer, glioblastoma, glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), hepatocellular cancer (HCC), kidney cancer, leukemia, acute myeloid leukemia (AML), lymphoma, diffuse large B cell lymphoma (DLBCL), lung cancer, non-small cell lung cancer (NSCLC), melanoma, advanced melanoma, uveal melanoma, metastatic melanoma, cutaneous melanoma, multiple myeloma, mesothelioma, nervous system-associated tumors, neuroblastoma, ovarian cancer, ovarian clear cell carcinomas, pancreatic cancer, pancreatic adenocarcinoma, prostate cancer, renal cell carcinoma, sarcoma, synovial sarcoma, soft tissue sarcoma, osteosarcoma, liposarcoma, or thymic carcinomas.

[0021] The details of various embodiments are set forth in the description below. Other features, objects and advantages will be apparent from the description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 provides a graph representing the protein expression of COL-1 mRNA.

[0023] Figure 2 provides graphs representing expression of some of the antigens in COL-1 mRNA.

[0024] Figure 3A provides a graph representing the mRNA formulations and the response against peptides. Figure 3B provides a graph representing the cytokine evaluation showing the CD8+ T cells results. Figure 3C provides a graph representing the cytokine evaluation showing the CD4+ T cells results.

[0025] Figure 4A provides a graph representing the mRNA formulations and the response against peptides. Figure 4B provides a graph representing the cytokine evaluation showing the CD8+ T cells results.

[0026] Figure 5A provides a graph representing the mRNA formulations and the response against peptides. Figure 5B provides a graph representing the cytokine evaluation showing the CD8+ T cells results. Figure 5C provides a graph representing the cytokine evaluation showing the CD4+ T cells results.

[0027] Figure 6A provides a graph representing the tumor volume for the buffer formulation group.

[0028] Figure 6B provides graphs representing the tumor volume and the survival percentage for a specific dose in the 3gp-LNP formulation group. Figure 6C provides a graph representing the tumorvolume for a specific dose in the 3gp-LNP formulation group. Figure 6D provides a graph representing the tumor volume for a specific dose in the 3gp-LNP formulation group.

[0029] Figure 7A provides a graph representing the tumor volume in the MC38gp tumor model. Figure 7B provides a graph representing the survival percentage over time in the MC38gp tumor model.

[0030] Figure 8A provides the results of the peripheral blood analysis from day 8 that was incubated with tetramers to gp33, gp34 or gp276.

[0031] Figure 8B provides the results of the tissue analysis from day 8 where the indicated tissues were collected, dissociated and incubated with tetramers to gp33 before analysis.

[0032] Figure 8C provides the results from the analysis of splenocytes that were collected on day 8 and restimulated with gp-peptides and the production of IFNy or TNFa was determined.

[0033] Figure 8D provides the splenocyte results from day 8 where the target cells were analyzed 4 hours after immunized animals were injected with gp33 -pulsed and CTV-labelled target cells intravenously.

[0034] Figure 9A provides the analysis of the peripheral blood from day 7 for gp33-specific CD8+ T cells.

[0035] Figure 9B shows the blood glucose measurement and diabetes incidence from the RIPgp animals evaluated.

[0036] Figure 10 provides the average tumor growth curves from the different treatment groups and the survival percentages with the treated animals and number of animals displaying a complete response (CRs) are indicated in parentheses.

[0037] Figure 11A provides the quantification of antigen-specific CD8+ T cells based on the analysis of the peripheral blood collected on day 11 which was incubated with gp33 tetramers.

[0038] Figure 1 IB provides the average tumor growth curves for the various treatment groups.

[0039] Figure 11C provides the survival percentages with the sample size and number of confirmed responders (CRs) indicated.

[0040] Figure 12A outlines the expression, represented as fold increase over buffer treated samples, of several inflammatory genes measured by qPCR in the muscle, dLN and spleen of C57BL / 6 mice at 6 and 24 hours after administration.

[0041] Figure 12B provides the expression results, shown relative to Actb, from the qPCR samples of the indicated genes in muscle tissue collected from the muscle tissue (injection site).

[0042] Figure 12C provides the expression results, shown relative to Actb, from the qPCR samples of the indicated genes in the draining inguinal lymph node (dLN).

[0043] Figure 12D provides the expression results, shown relative to Actb, from the qPCR samples of the indicated genes in the spleen.

[0044] Figure 12E provides the expression of several inflammatory genes measured by qPCR of C57BL / 6 mice at 6 and 24 hours after administration as well as the serum protein expression of the indicated cytokines or chemokines.

[0045] Figure 12F provides the expression of IFN genes in the muscle at 6 and 24 hours.

[0046] Figure 12G provides the serum IFNa and IFNP protein levels at 6 and 24 hours.

[0047] Figure 12H provides the frequency of pDCs (SiglecFFCD 1 lcmid) in the blood and spleen as measured on days 1 or 7.

[0048] Figure 13A shows the blood glucose measurements from RlPgp animals for each group, with each line representing an individual animal (mouse).

[0049] Figure 13B shows the diabetes incidence from the RlPgp animals and the number of responders.

[0050] Figure 13C provides the expression of the specified genes in muscle, relative to Actb, for the different treatment groups.

[0051] Figure 13D provides the average tumor growth and survival percentages for the treatment groups.

[0052] Figure 13E provides the frequency of the neutrophils (Ly6G+CDl lb+) found in the spleen, blood and tumor.

[0053] Figure 13F provides the expression of CXCL2 in the tumor, spleen, and blood.

[0054] Figure 13G provides the expression of CXCL2 in the tumor tissue quantified by qTR-PCR and expressed relative to Actb.

[0055] Figure 13H provides the frequency of gp33-specific CD8+ T cells in the spleen, blood, draining inguinal LN (dLN) and tumor.

[0056] Figure 131 provides the percentage of 4-1BB expression on gp33-specific CD8+ T cells in the referenced tissues.

[0057] Figure 13J provides flow cytometry plots of gp33-specific CD8+ T cells collected from the MC38gp tumor.

[0058] Figure 14 provides the neoantigen-specific T cell responses after vaccine administration.

[0059] Figure 15A provides a graph representing the survival after administration with mRNA encoding EGFRvIII.

[0060] Figure 15B provides images showing the effect of administration with mRNA encoding EGFRvIII.DETAILED DESCRIPTION

[0061] The following description sets forth exemplary compounds, compositions, methods, parameters and the like. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure but is instead provided as a description of exemplary embodiments.

[0062] The delivery of a therapeutic agent to a subject is important for its therapeutic effects and usually it can be impeded by limited ability of the compound to reach targeted cells and tissues.Improvement of such therapeutic agents to enter the targeted cells of tissues by a variety of means of delivery is crucial. Nucleic acid therapy has emerged as the dominant method of treating various diseases and therapeutic indications given the versatility, lower immune response and higher potency as compared to traditional therapies.I, COMPOSITIONS OF PRESENT DISCLOSURE

[0063] Described herein are polynucleotides (e.g., mRNAs), compositions, formulations, methods, and / or use of nucleic acid vaccines, specifically nucleic acid vaccines comprising polynucleotides encoding one or more proteins, polypeptides or peptides fragments or variants thereof of tumor-associated antigens for the prevention, alleviation and / or treatment of diseases, disorders and / or conditions caused by tumor-associated antigens.Tumor-Associated Antigens

[0064] Antigens where expression has been associated with embryonic development, in adult mammals, to the placenta and testis germline cells and cancerous tissues were selected as the starting point to select sequences for the nucleic acid vaccines described herein. The selection of these antigens was based on many aspects including, but not limited to: (1) immunogenicity of a given target, (2) the effectiveness the antigen is processed and loaded into MHC molecules in normal tissues, (3) level of accessibility and immunosurveillance of normal tissues expressing low levels of the selected antigen, and (4) availability of beneficial safety profiles of immunotherapeutic modalities targeting one or more of the selected antigens.

[0065] In some embodiments, the tumor-associated antigens described herein are characterized across multiple tumor types for inducing an anti -tumor immune response. As a non-limiting example, the tumor-associated antigens show restricted expression in normal tissue but high immunogenicity and high prevalence in one or more tumor tissue of interest.

[0066] In some embodiment, the tumor-associated antigens described herein are used in combination in a nucleic acid vaccine to cover multiple tumor types or antigens for a particular tumor in a subject. While not wishing to be bound by theory, combining antigens in a vaccine could lead to an additive orsynergistic effect to a subject especially if a tumor expresses more than one antigen. Additionally, a vaccine encoding more than one antigen supports a broader coverage of eligible subjects and tumors that can be treated with the vaccine due to the mosaic expression provided by the combination of antigens in the vaccine. Normally, this combination approach or personalized approach to achieve a broach coverage for a subject is cost prohibitive from a manufacturing and a regulatory perspective. However, by utilizing antigens characterized across multiple tumor types, then selecting and optimizing critical sequences from these antigens to create nucleic acid vaccines to provide broad coverage across tumor types and subjects, a personalized vaccine is able to provide immunogenicity across multiple tumor types in for different subjects.

[0067] In some embodiments, the polynucleotide may be designed to encode one or more polypeptides of interest from tumor-associated antigens or fragments or variants thereof. The tumor-associated antigens described herein may be associated with various malignancies such as, but not limited to, melanoma, ovarian cancer lung cancer, synovial sarcoma, head and neck cancer, solid tumors, and / or glioblastoma multiforme (GBM). Such polypeptide of interest of tumor-associated antigens may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides or variants of polypeptides, which independently may be encoded by one or more regions or parts or the whole of a polynucleotide from any of MAGE-A4, MAGE-A3, NY-ESO-1, PRAME, SSX, TTK, SPA17, and / or EGFRvIII. As used herein, the term “polypeptides of interest” refers to any polypeptide which is selected to be encoded within, or whose function is affected by, the polynucleotides described herein. Melanoma-Associated Antigen 3 (MAGE-A3) and Melanoma-Associated Antigen 4 (MAGE-A4)

[0068] Melanoma- Associated Antigen 4 (MAGE-A4) and Melanoma-Associated Antigen 3 (MAGE- A3) are members of the MAGE-A gene family which are tumor-specific antigens. MAGE-A4 is a protein coding gene which is generally known to regulate cell proliferation through inhibition of cell cycle arrest at the G1 phase and MAGE-A4 also negatively regulates p53-mediated apoptosis. MAGE-A3 is a protein coding gene which is generally known to be an activator of ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin-protein ligases that acts as a repressor of autophagy, and MAGE-A3 may play a role in embryonal development and tumor transformation or aspects of tumor progression.

[0069] While MAGE-A4 and MAGE-A3 is expressed in a range of epithelial malignancies, MAGE-A4 is almost expressed by synovial sarcomas but it is also expressed in non-small cell lung cancer and MAGE-A3 is often associated with melanoma and ocular melanoma but it is also expressed in lung cancer including non-small cell lung cancer, breast cancer, head and neck cancer, bladder cancer, ovarian cancer and renal cell carcinoma.New York Esophageal Squamous Cell Carcinoma 1 (NY-ESO-1)

[0070] New York Esophageal Squamous Cell Carcinoma 1 (NY -ESO-1) which is also known as cancer / testis antigen IB (CTAG1B) is limited to germ cells but is frequently identified in cancer cells is silenced in normal somatic cells except for male testis. However, NY-ESO-1 is expressed in many cancer cells as a consequence of what is assumed in the art as an epigenetic event but the molecular mechanisms of NY-ESO-1 in tumorigenesis and cancer dissemination remains unknown. Generally, NY-ESO-1 is a protein coding gene which is generally known to be highly immunogenic and can trigger spontaneous humoral and cellular immune responses. NY-ESO-1 is expressed in a number of tumors, including esophageal, lung, hepatocellular, prostate, gastric, colorectal, and breast cancer. It is also expressed in melanomas including advanced myelomas where it is associated with tumor proliferation.Preferentially Expressed Antigen in Melanoma (PRAME)

[0071] Preferentially Expressed Antigen in Melanoma (PRAME) is not expressed in normal tissues, except testis. PRAME is a protein coding gene and is generally believed to confer a growth advantage to cancer cells as PRAME acts as a repressor of retinoic acid receptor. PRAME is expressed in certain types of cancer cells including melanoma (e.g., uveal melanoma, and metastatic melanoma), sarcoma, synovial sarcoma, non-small cell lung cancer, neuroblastoma, leukemia, lymphoma, female reproductive system-associated carcinomas (e.g., endometrial and uterine serous carcinomas), ovarian clear cell carcinomas, thymic carcinomas, and basal cell carcinomas.Synovial Sarcoma X Family Member 2 (SSX2)

[0072] Synovial Sarcoma, X (SSX) Family Member 2 (SSX2) may function as transcriptional repressors and are capable of eliciting spontaneous humoral and cellular immune responses in cancer patients. SSX2, and other family members SSX1 and SSX4, have been involved in translocations that are characteristically found in synovial sarcomas. Diseases associated with SSX2 include synovial sarcoma, soft tissue sarcoma, melanoma, head and neck squamous cell carcinoma (HNSCC), breast cancer, pancreatic adenocarcinoma, but also can be found in AML and cholangiocarcinoma.Threonine Tyrosine Kinase (TTK)

[0073] Threonine Tyrosine Kinase (TTK; may also be referred to as Dual specificity protein kinase TTK) is high in proliferating cells and tissues and low or even absent in resting cells. TTK is a dualspecificity protein kinase that regulates chromosome alignment and segregation during mitosis and is also involved in kinetochore localization and the spindle assembly checkpoint. Diseases associated with TTK include pancreatic cancer, lung cancer, melanoma, ovarian cancer, non-small cell lung cancer (NSCLC), esophageal cancer and breast cancer, multiple myeloma, osteosarcoma, hepatocellular carcinoma, neuroblastoma, glioma, glioblastoma, mesothelioma, prostate cancer, colon cancer, endometrial cancer, bladder cancer, and head and neck cancer.Sperm Autoantigenic Protein 17 (SPA 17)

[0074] Sperm Autoantigenic Protein 17 (SPA 17) may be involved in cell-cell adhesion such as immune cell migration and metastasis. While not wishing to be bound by theory, the SPA 17 protein has been characterized in the literature by its involvement in the binding of sperm to the zona pellucida of the oocyte. Diseases associated with SPA 17 include multiple myeloma, diffuse large B cell lymphoma (DLBCL), dedifferentiated liposarcoma, endometrial, ovarian, and cervical cancers, nervous system-associated tumors, esophageal, breast, and lung cancers, as well as kidney, bladder, and prostate malignancies.Epidermal Growth Factor Receptor Variant III (EGFRvIII)

[0075] Epidermal growth factor receptor (EGFR) is a gene that makes a protein that is involved in cell growth and cell survival. Variants of EGFR protein have been identified in different forms of cancer and have been theorized to help the cancer cells grow and spread in the body of a subject. One of the variants of EGFR that has been identified to be associated with different forms of cancer is EGFR variant III (EGFRvIII). EGFRvIII has been associated with glioblastoma (GBM) which is a brain tumor as well as other brain cancers, head and neck cancers, ovarian cancer, prostate cancers, lung cancer, and breast cancers.Polynucleotides for Nucleic Acid Vaccines

[0076] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein encode one of the tumor-associated antigens, or a fragment or a of the variant of the tumor-associated antigens described herein.

[0077] In some embodiments, the nucleic acid vaccine described herein may encode one or more proteins, polypeptides, peptides, fragments or variants thereof of tumor-associated antigens or a combination thereof. Non-limiting examples of sequences of the tumor-associated antigens which can be used to develop the nucleic acid vaccines are provided in Table 1.

[0078] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigens MAGE-A3 and MAGE-A4, or a fragment, or a variant of a region of the tumor-associated antigens MAGE-A3 and MAGE-A4.

[0079] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen NY-ESO-1, or a fragment, or a region of the tumor-associated antigen NY-ESO-1.

[0080] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen PRAME, or a fragment, or a region of the tumor-associated antigen PRAME.

[0081] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen SSX2, or a fragment, or a region of the tumor-associated antigen SSX2.

[0082] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen TTK, or a fragment, or aregion of the tumor-associated antigen TTK.

[0083] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen SPA 17, or a fragment, or a region of the tumor-associated antigen SPA 17.

[0084] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises a sequence encoding a region of the tumor-associated antigen EGFRvIII, or a fragment, or a region of the tumor-associated EGFRvIII.

[0085] In some embodiments, the polynucleotides of the nucleic acid vaccine described herein comprises sequence regions so that the nucleic acid vaccine encodes more than one tumor-associated antigen. As a non-limiting example, the nucleic acid vaccine can encode MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX2, TTK, SPA17 or a combination thereof of each of these tumor-associated antigens. As another non-limit example, the nucleic acid vaccine can encode EGFRvIII.Table 1. Tumor- Associated Antigens

[0086] In some embodiments, the nucleic acid vaccine described herein may encode at least one of the tumor-associated antigen amino acid sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include both an antigen sequence region and an epitope sequence region for a tumor-associated antigen, which may be the same or different tumor-associated antigen. In some embodiments, the nucleic acid vaccine described herein may encode tumor-associated antigens, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 of the tumor-associated antigen.

[0087] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes MAGE-A3 and MAGE-A4 antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding MAGE-A3 and MAGE-A4 and an epitope sequence region encoding MAGE-A3 and MAGE-A4. In some embodiments, the nucleic acid vaccine described herein may encode MAGE-A3 and MAGE-4 antigen, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for MAGE-A3 and / or MAGE-A4.

[0088] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes NY-ESO-1 antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding NY-ESO-1 and an epitope sequence region encoding NY-ESO-1. In some embodiments, the nucleic acid vaccine described herein may encode the NY-ESO-1 antigen, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for NY-ESO-1.

[0089] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes PRAME antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding PRAME and an epitope sequence region encoding PRAME. In some embodiments, the nucleic acid vaccine described herein may encode the PRAME antigen, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for PRAME.

[0090] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes SSX2 antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding SSX2 and an epitope sequence region encoding SSX2. In some embodiments, the nucleic acid vaccine described herein may encode the SSX2 antigen, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for SSX2.

[0091] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes TTK antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding TTK and an epitope sequence region encoding TTK. In some embodiments, the nucleic acid vaccine described herein may encode the TTK antigen, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for TTK.

[0092] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes SPA17 antigen with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include an antigen sequence region encoding SPA 17 and an epitope sequence region encoding SPA17. In some embodiments, the nucleic acid vaccine described herein may encode the SPA 17 antigen, where the nucleic acid vaccine comprises sequences or encodessequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for SPA17.

[0093] In some embodiments, the nucleic acid vaccine described herein may comprise a sequence which encodes EGFRvIII with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of any of the sequences in Table 1 or fragments of any of the sequences in Table 1 or variants of any of the sequences in Table 1. In one embodiment, the coding sequences of mRNA vaccines described herein may include a sequence region encoding EGFRvIII. In some embodiments, the nucleic acid vaccine described herein may encode EGFRvIII, where the nucleic acid vaccine comprises sequences or encodes sequences with at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or fragment thereof in Table 1 for EGFRvIII.

[0094] In some embodiments, the nucleic acid vaccine may be an mRNA vaccine that, when translated, produces one or more proteins, peptides, fragments or variants thereof of the tumor-associated antigens. Accordingly, the polynucleotides of the mRNA vaccine are mRNA polynucleotides encoding one or more proteins, polypeptides, peptides, fragments or variants thereof of the tumor-associated antigens.

[0095] Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by a polynucleotide described herein, any of several manipulations and / or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating. Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules described herein. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full-length molecule would.

[0096] In some embodiments, the nucleic acid vaccines described herein comprise an mRNA polynucleotide encoding one or more proteins, polypeptides, or peptides, fragments or variants of the tumor-associated antigens such as, but not limited to, those in Table 1.

[0097] Non-limiting examples of a RNA sequence encoding proteins, peptides, fragments or variants of the tumor-associated antigens are provided in Table 2.Table 2. Sequences of Tumor-Associated Antigens

[0098] In some embodiments, the mRNA polynucleotide encoding tumor-associated antigens comprises the coding sequence of SEQ ID NO. 21-53, or a variant thereof.

[0099] In some embodiments, the mRNA polynucleotide encoding tumor-associated antigens or variants thereof comprises the coding sequence of SEQ ID NO. 166-185, or a variant thereof.

[0100] In some embodiments, the mRNA polynucleotide encoding tumor-associated antigens or variant thereof comprises SEQ ID NO. 37. The mRNA polynucleotide encodes six partial tumor-associated antigen sequences and seven characterized HLA-restricted epitopes derived from the tumor-associated antigens MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX2, TTK, and SPA17.

[0101] In some embodiments, the nucleic acid vaccines may comprise a region encoding any of the sequences listed in Tables 1 or a fragment or peptide or variant thereof. The nucleic acid vaccines may comprise hybrid or chimeric regions, or mimics or variants. In some embodiments, the nucleic acid vaccines may comprise any of the polynucleotide sequences listed in Table 2-3.Table 3. Exemplary Sequences to be Used in the Nucleic Acid Vaccines

[0102] Any of the sequences referred to in Tables 1-3 or variants thereof may also be used in a booster vaccine described herein. In some embodiments, any of the sequences referred to in Tables 1-3 or variants thereof may also be used in a booster vaccine shortly after the identification or re-identification of any of the cancer, tumor, symptoms or conditions in a subject.

[0103] In some embodiments, the nucleic acid vaccine described herein encodes tumor-associated antigen or a region of a tumor-associated antigen or fragment or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a protein provided by an amino acid sequence in Table 1. In some embodiments, the nucleic acid vaccine described herein encodes a protein or fragment or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a tumor-associated antigen or a region of a tumor-associated antigen provided by an amino acid sequence in Table1. The terms “identical” or percent “identity” in the context of two or more polypeptide sequences refer to two or more sequences that are the same. The percent identity between polypeptide sequences may be performed using algorithms known in the art, such as BLAST and CLUSTAL.

[0104] The sequence of the tumor-associated antigen protein, fragment or peptide or variant thereof may be obtained from any source. In some embodiments, the sequence of the tumor-associated antigen protein or fragment or peptide or variant thereof is from a tumor that has restricted expression in normal tissue and high prevalence cancerous or non-normal tissue.

[0105] In some embodiments, the polynucleotide sequence of the tumor-associated antigen protein, or a fragment or peptide or variant thereof may be modified or optimized (such as codon optimized) for expression in a particular cell or host organism.

[0106] In some embodiments, the nucleic acid vaccine described herein may be a multivalent vaccine. The multivalent vaccine may include polynucleotides that encodes at least two different tumor-associated antigen proteins, polypeptides, peptides, fragments or variants thereof. As a non-limiting example, the polynucleotides may encode an antigen and epitope from MAGE-A3 / MAGE-A4, NY-ESO-1, PRAME, SSX2, TTK, and / or SPA17. As a non-limiting example, the polynucleotides may encode a peptide or fragment from an EGFRvIII variant.

[0107] In some embodiments, the nucleic acid vaccine is a multivalent vaccine and MAGE-A3 / MAGE-A4 (MAGE-A3 / MAGE-A4 antigen and MAGE-A4 epitope), NY-ESO-1 (antigen and epitope from NY-ESO-1), PRAME (antigen and epitopes from PRAME), SSX2 (antigen and epitope from SSX2), TTK (antigen and epitope from TTK) and SPA 17 (antigen and epitope from SPA 17). As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 21-33, 34-41, and / or 46-53 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 21 and 22 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 23 and 24 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 25, 26 and 27 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 28 and 29 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 30 and 31 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 32 and 33 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 34 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 35 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 36 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 37 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 38 or a fragment or variant thereof. As a non-limitingexample, the nucleic acid vaccine may include SEQ ID NO. 39 or a fragment or variant thereof. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 40 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 41 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 46 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 47 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 48 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 49 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 50 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 51 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 52 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 53 or a fragment or variant thereof. Any of SEQ ID NO. 21-33, 34-41, and / or 46-53 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0108] In some embodiments, the nucleic acid vaccine is a multivalent vaccine and MAGE-A3 / MAGE-A4 (MAGE-A3 / MAGE-A4 antigen and MAGE-A4 epitope), NY-ESO-1 (antigen and epitope from NY-ESO-1), PRAME (antigen and epitopes from PRAME), SSX2 (antigen and epitope from SSX2), and SPA17 (antigen and epitope from SPA17). As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 21-29, 32, 33, 42, 43, 44, and / or 45 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 21 and 22 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 23 and 24 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 25, 26 and 27 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 28 and 29 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 32 and 33 or a fragment or variant thereof. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 42 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 43 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 44 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 45 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 53 or a fragment or variant thereof. Any of SEQ ID NO. 21-29, 32, 33, 42, 43, 44, and / or 45 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0109] In some embodiments, the nucleic acid vaccine comprises a sequence encoding the tumor-associated antigens MAGE-A3 and MAGE-A4 in one amino acid sequence (SEQ ID NO. 3) instead of two separate sequences. Additionally, the nucleic acid vaccine comprises a sequence encoding the MAGE-A4 epitope (SEQ ID NO. 4). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for MAGE-A3 and MAGE-A4 (SEQ ID NO. 54 for DNA and SEQ ID NO. 21 for RNA) and comprises a sequence encoding MAGE-4 epitope (SEQ ID NO. 55 for DNA and SEQ ID NO. 22 for RNA).

[0110] In some embodiments, the nucleic acid vaccine comprises a sequence encoding NY-ESO-1 partial antigen (SEQ ID NO. 6) and comprises a sequence encoding NY-ESO-1 epitope (SEQ ID NO. 7). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for NY-ESO-1 (SEQ ID NO. 56 for DNA and SEQ ID NO. 23 for RNA) and comprises a sequence encoding NY-ESO-1 epitope (SEQ ID NO. 57 for DNA and SEQ ID NO. 24 for RNA).[oni] In some embodiments, the nucleic acid vaccine comprises a sequence encoding PRAME partial antigen (SEQ ID NO. 9) and comprises a sequence encoding PRAME epitope (SEQ ID NO. 10-11). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for PRAME (SEQ ID NO. 58 for DNA and SEQ ID NO. 25 for RNA) and comprises a sequence encoding first PRAME epitope (SEQ ID NO. 59 for DNA and SEQ ID NO. 26 for RNA) and a sequence encoding second PRAME epitope (SEQ ID NO. 60 for DNA and SEQ ID NO. 27 for RNA).

[0112] In some embodiments, the nucleic acid vaccine comprises a sequence encoding SSX2 partial antigen (SEQ ID NO. 13) and comprises a sequence encoding SSX2 epitope (SEQ ID NO. 14). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for SSX2 (SEQ ID NO. 61 for DNA and SEQ ID NO. 28 for RNA) and comprises a sequence encoding SSX2 epitope (SEQ ID NO. 62 for DNA and SEQ ID NO. 29 for RNA).

[0113] In some embodiments, the nucleic acid vaccine comprises a sequence encoding TTK partial antigen (SEQ ID NO. 16) and comprises a sequence encoding TTK epitope (SEQ ID NO. 17). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for TTK (SEQ ID NO. 63 for DNA and SEQ ID NO. 30 for RNA) and comprises a sequence encoding TTK epitope (SEQ ID NO. 64 for DNA and SEQ ID NO. 31 for RNA).

[0114] In some embodiments, the nucleic acid vaccine comprises a sequence encoding SPA 17 partial antigen (SEQ ID NO. 19) and comprises a sequence encoding SPA17 epitope (SEQ ID NO. 20). In some embodiments, the nucleic acid vaccine comprises a sequence encoding a partial antigen for SPA 17 (SEQ ID NO. 65 for DNA and SEQ ID NO. 32 for RNA) and comprises a sequence encoding SPA17 epitope (SEQ ID NO. 66 for DNA and SEQ ID NO. 33 for RNA).1

[0115] In some embodiments, the nucleic acid vaccine encodes a human IgK signal peptide (SEQ ID NO. 87 for amino acid; SEQ ID NO. 88 for DNA; SEQ ID NO. 89 for RNA).

[0116] Cleavage sequences, including cathepsin cleavage sequences, are well known in the art and can be used for many purposed including, but not limited to, help to pre-process smaller sequences from a larger sequence by facilitating the cleavage of each element from the full-length, may help target the amino acid / protein toward the lysosome, and / or may alter the tertiary protein structure. In some embodiments, the nucleic acid vaccine comprises cleavage sequences to separate the tumor-associate antigen sequences if the nucleic acid vaccine is a multivalent vaccine. Cleavage sequences are known in the art, however, non-limiting examples of cleavage sequences that can be used with the nucleic acid vaccines include SEQ ID NO. 90 (91-102 for DNA, 103-114 for RNA) for 7 amino acid cleavage sequence and SEQ ID NO. 115 (116-127 for DNA, 128-139 for RNA) for 12 amino acid cleavage sequence.

[0117] In some embodiments, the nucleic acid vaccine may comprise additional regions to the sequence such as, but not limited to, a sequence tag. For example, sequence tags or amino acids, such as one or more lysines, can be added (e.g., at the N-terminal or C-terminal ends) which can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Sequence tags are known in the art, however, a non-limiting example of sequence tags that can be used with the nucleic acid vaccine include SEQ ID NO. 140, 143, 146 (SEQ ID NO. 141, 144, 147 for DNA; SEQ ID NO. 144, 145, 148 for RNA).

[0118] In some embodiments, the nucleic acid vaccine encodes tumor-associated antigens MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX-2, TTK, and SPA17, where the signal peptide is human IgG kappa light chain signal peptide, and where the tumor-associated antigen sequences are separated by cleavage sequences. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 67, 68, 69, 70, 71, 72, 73, and / or 74 (34, 35, 36, 37, 38, 39, 40, and / or 41 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 67 (34 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 68 (35 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 69 (36 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 70 (37 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 71 (38 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 72 (39 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 73 (40 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 74 (41 for RNA) or a fragment or variantthereof. Any of SEQ ID NO. 34, 35, 36, 37, 38, 39, 40, and / or 41 may be fully modified with Nl-methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0119] In some embodiments, the nucleic acid vaccine encodes tumor-associated antigens MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX-2, and SPA17, where the signal peptide is human IgG kappa light chain signal peptide, and where the tumor-associated antigen sequences are separated by cleavage sequences. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 75, 76, 77, and / or 78 (SEQ ID NO. 42, 43, 44 and / or 45 for RNA) or a fragment or variant thereof. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 75 (42 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 76 (43 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 77 (44 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 78 (45 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 42, 43, 44 and / or 45 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0120] In some embodiments, the nucleic acid vaccine encodes tumor-associated antigens MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX-2, TTK, and SPA17, where the signal peptide is human IgG kappa light chain signal peptide and where the nucleic acid vaccine includes a sequence tag. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 79, 80, 81, and / or 82 (SEQ ID NO.46, 47, 48, and / or 49 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 79 (46 for RNA) or a fragment or variant thereof. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 80 (47 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 81 (48 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 82 (49 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 46, 47, 48, and / or 49 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0121] In some embodiments, the nucleic acid vaccine encodes tumor-associated antigens MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX-2, TTK, and SPA17, where the tumor-associated antigen sequences are separated by cleavage sequences, where the signal peptide is human IgG kappa light chain signal peptide and where the nucleic acid vaccine includes a sequence tag. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 83, 84, 85, and / or 86 (SEQ ID NO. 50, 51, 52 and / or 53 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 83 (50 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 84 (51 for RNA) or a fragment or variant thereof. As anon-limiting example, the nucleic acid vaccine may include SEQ ID NO. 85 (52 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 86 (53 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 50, 51, 52 and / or 53 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0122] In some embodiments, the nucleic acid vaccine encodes an EGFRvIII variant. As a nonlimiting example, the nucleic acid vaccine may include SEQ ID NO. 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, and / or 185 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 166 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 167 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 168 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 169 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 170 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 171 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 172 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 173 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 174 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 175 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 176 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 177 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 178 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 179 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 180 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 181 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 182 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 183 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 184 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 185 or a fragment or variant thereof.

[0123] In some embodiments, the nucleic acid vaccine encodes EGFRvIII of SEQ ID NO. 161 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 186, 191, 192, and / or 193 (166, 171, 172 and / or 173 for RNA) or a fragment or variant thereof. As anon-limiting example, the nucleic acid vaccine may include SEQ ID NO. 186 (166 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 191 (171 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 192 (172 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 193 (173 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 186, 191, 192, 193, 166, 171, 172, and / or 173 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0124] In some embodiments, the nucleic acid vaccine encodes EGFRvIII of SEQ ID NO. 162 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 187, 194, 195, and / or 196 (167, 174, 175 and / or 176 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 187 (167 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 194 (174 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 195 (174 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 196 (176 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 187, 194, 195, 196, 167, 174, 175, and / or 176 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0125] In some embodiments, the nucleic acid vaccine encodes EGFRvIII of SEQ ID NO. 163 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 188, 197, 198, and / or 199 (168, 177, 178 and / or 179 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 188 (168 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 197 (177 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 198 (178 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 199 (179 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 188, 197, 198, 199, 168, 177, 178, and / or 179 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0126] In some embodiments, the nucleic acid vaccine encodes EGFRvIII of SEQ ID NO. 164 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 189, 200, 201, and / or 202 (169, 180, 181 and / or 182 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 189 (169 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 200 (180 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 201 (181 for RNA) or a fragment or variant thereof. As a non-limitingexample, the nucleic acid vaccine may include SEQ ID NO. 202 (182 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 189, 200, 201, 202, 169, 180, 181, and / or 182 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0127] In some embodiments, the nucleic acid vaccine encodes EGFRvIII of SEQ ID NO. 165 or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 190, 203, 204, and / or 205 (170, 183, 184 and / or 185 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 190 (170 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 203 (183 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 204 (184 for RNA) or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine may include SEQ ID NO. 205 (185 for RNA) or a fragment or variant thereof. Any of SEQ ID NO. 190, 203, 204, 205, 170, 183, 184, and / or 185 may be fully modified with N1 -methylpseudouridine if they are used or incorporated into an mRNA or in a nucleic acid vaccine.

[0128] In some embodiments, the nucleic acid vaccine encodes the tumor-associated antigens described herein where the nucleic acid vaccine may include SEQ ID NO. 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, or 185 or a fragment or variant thereof and at least one stop codon. The stop codon or stop codons may be any known in the art such as, but not limited to, the stop codons described herein. As a non-limiting example, the nucleic acid vaccine comprises one stop codon. As a non-limiting example, the nucleic acid vaccine comprises two stop codons. As a non-limiting example, the nucleic acid vaccine comprises two stop codons selected from UGA, UAA, UGA or UAG. As a nonlimiting example, the nucleic acid vaccine comprises two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 34 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 34 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 34 and two stop codons, wherein the two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 35 and two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 36 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 37 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.38 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 38 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.38 and two stop codons, wherein the two stop codons are UAAUGA. As a non-limiting example, thenucleic acid vaccine comprises SEQ ID NO. 39 and two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 40 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.41 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 42 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 42 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 42 and two stop codons, wherein the two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 43 and two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 44 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 45 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 46 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 46 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 46 and two stop codons, wherein the two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 47 and two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 48 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 49 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 50 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 50 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 50 and two stop codons, wherein the two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 51 and two stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 52 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 53 which comprises two stop codons, and the stop codons are UAAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 173 and at least one stop codon. As anon-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 173 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 173 and two stop codons, wherein the two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 166 and two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 172 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 171 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleicacid vaccine comprises SEQ ID NO. 176 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 176 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 176 and two stop codons, wherein the two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 167 and two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 175 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 174 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 179 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 179 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 179 and two stop codons, wherein the two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 168 and two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 178 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 177 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 182 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 182 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 182 and two stop codons, wherein the two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 169 and two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 181 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 180 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 185 and at least one stop codon. As anon-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 185 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 185 and two stop codons, wherein the two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 170 and two stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 184 which comprises two stop codons, and the stop codons are UGAUGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 183 which comprises two stop codons, and the stop codons are UGAUGA.

[0129] In some embodiments, the nucleic acid vaccine encodes the tumor-associated antigens described herein where the nucleic acid vaccine may include SEQ ID NO. 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 , or 205 or a fragment or variant thereof and at least one stop codon.The stop codon or stop codons may be known in the art such as, but not limited to, the stop codons described herein. As a non-limiting example, the nucleic acid vaccine comprises one stop codon. As a non-limiting example, the nucleic acid vaccine comprises two stop codons. As a non-limiting example, the nucleic acid vaccine comprises two stop codons selected from TGA, TAA, TGA or TAG. As a nonlimiting example, the nucleic acid vaccine comprises two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 67 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 67 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 67 and two stop codons, wherein the two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 68 and two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 69 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 70 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.71 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 71 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.71 and two stop codons, wherein the two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 72 and two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 73 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO.74 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 75 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 75 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 75 and two stop codons, wherein the two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 76 and two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 77 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 78 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 79 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 79 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 79 and two stop codons, wherein the two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 80 and two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 81 which comprises two stop codons, and the stop codons areTAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 82 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 83 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 83 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 83 and two stop codons, wherein the two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 84 and two stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 85 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 86 which comprises two stop codons, and the stop codons are TAATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 193 and at least one stop codon. As anon-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 193 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 193 and two stop codons, wherein the two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 186 and two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 192 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 191 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 196 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 196 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 196 and two stop codons, wherein the two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 187 and two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 195 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 194 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 199 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 199 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 199 and two stop codons, wherein the two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 188 and two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 198 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 197 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 202 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 202 and two stop codons. As a non-limiting example, thenucleic acid vaccine comprises SEQ ID NO. 202 and two stop codons, wherein the two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 189 and two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 201 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 200 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 205 and at least one stop codon. As anon-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 205 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 205 and two stop codons, wherein the two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 190 and two stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 204 which comprises two stop codons, and the stop codons are TGATGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO. 203 which comprises two stop codons, and the stop codons are TGATGA.Components of Nucleic Acid Vaccines

[0130] The term “nucleic acid,” in its broadest sense, includes any compound and / or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a -D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA) or hybrids or combinations thereof.

[0131] Ribonucleic acid (RNA) is a molecule that is made up of nucleotides, which are ribose sugars attached to nitrogenous bases and phosphate groups. The nitrogenous bases include adenine (A), guanine (G), uracil (U), and cytosine (C). Generally, RNA mostly exists in the single-stranded form but can also exists double-stranded in certain circumstances. The length, form and structure of RNA is diverse depending on the purpose of the RNA. For example, the length of an RNA can vary from a short sequence (e.g., siRNA) to a long sequence (e.g., IncRNA), can be linear (e.g., mRNA) or circular (e.g., circRNA), and can either be a coding (e.g., mRNA) or a non-coding (e.g., IncRNA) sequence.

[0132] In some embodiments, in vitro transcription (IVT) enzymatic synthesis methods may be used to make the polynucleotides of the nucleic acid vaccines described in the present disclosure.

[0133] In some embodiment, the nucleic acid vaccines described herein may include regions which differ in size and / or encoded protein. As non-limiting examples, the polynucleotide of the presentdisclosure may comprise a region encoding a heterogeneous signal peptide. As another non-limiting example, the polynucleotide of the present disclosure may comprise a non-coding polynucleotide.

[0134] In some embodiments, the length of a region encoding at least one peptide or polypeptide of interest of the polynucleotides of the nucleic acid vaccine is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, or up to and including 7,000 nucleotides). As used herein, such a region may be referred to as a “coding region” or a “region encoding.”

[0135] In some embodiments, the polynucleotides of the nucleic acid vaccine is or functions as a messenger RNA (mRNA). As used herein, the term “messenger RNA (mRNA)” refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide or polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[0136] The shortest length of a region of the polynucleotide of the nucleic acid vaccine can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g., no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.

[0137] The region of the polynucleotide of the nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants thereof described herein may be greater than about 30 nucleotides in length. The length may be, but is not limited to, at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 4,000, 5,000, 6,000, or up to and including 7,000, nucleotides. In some embodiments, the region includes from about 30 to about 7,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000 nucleotides).mRNA Components

[0138] The nucleic acid vaccines described herein may be an mRNA vaccine. The mRNA vaccine includes at least one mRNA molecule which, when translated, produce at least one peptide or polypeptide of interest for the prevention, alleviation and / or treatment of diseases, disorders and / or conditions cause by tumor-associated antigens. In general, an mRNA molecule generally includes at least a coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. In some aspects, one or more structural and / or chemical modifications or alterations may be included in the RNA which can reduce the innate immune response of a cell in which the mRNA is introduced.mRNA Components: Start Codon and Stop Codon

[0139] In some embodiments, the mRNA vaccine includes a region to initiate translation. This region may include any translation initiation sequence or signal including a start codon. As a non-limiting example, the region includes a start codon. In some embodiments, the start codon may be selected from, but not limited to, ATG, ACG, AGG, ATA, ATT, CTG, GTG, TTG, AUG, AUA, AUU, CUG, GUG, or UUG.

[0140] In some embodiments, the mRNA vaccine comprises a region comprising a start codon. The region comprising the start codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0141] In some embodiments, the mRNA vaccine includes a region to stop translation. This region may include any translation termination sequence or signal including at least one stop codon. As a nonlimiting example, the region includes a stop codon. As a non-limiting example, the region includes two stop codons. In some embodiments, the stop codon may be selected from, but not limited to, TGA, TAA, TGA, TAG, UGA, UAA, UGA or UAG.

[0142] In some embodiments, the mRNA vaccine includes the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA.

[0143] In some embodiments, the mRNA vaccine comprises a region comprising a stop codon. The region comprising the stop codon may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length.

[0144] In some embodiments, the regions to initiate or terminate translation may independently range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. Additionally, these regions may comprise one or more signal and / or restriction sequences.

[0145] In some embodiments, an agent may be used to mask a first start codon or non-preferred start codon in order to increase the chance that translation will initiate on a second start codon or preferred start codon downstream to the masked first start codon or non-preferred start codon.mRNA Components: Coding Region

[0146] In some embodiments, the coding region of the polynucleotide of the nucleic acid vaccine may encode at least one peptide or polypeptide of interest. Non-limiting examples of peptides or polypeptides of interest include one or more proteins, polypeptides, peptides, fragments or variants thereof of tumor-associated antigens for the prevention, alleviation and / or treatment of diseases, disorders and / or conditions caused by tumor-associated antigens.mRNA Components: Untranslated Region

[0147] The polynucleotides of the nucleic acid vaccines described herein may comprise one or more regions or parts which act or function as an untranslated region (UTR). Wild type UTRs of a gene are transcribed but not translated. In mRNA, the 5 'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. While not wishing to be bound by theory, UTRs may have a role in the stability and translation of the nucleic acid molecule. Variants of UTRs may be utilized where one or more nucleotides (e.g., A, T / U, C or G) are added or removed to the termini of the UTR.

[0148] In some embodiments, the UTRs of the polynucleotide of the nucleic acid vaccine may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0149] Wild type 5' UTRs include features which play roles in translation initiation as these 5’ UTRs include sequences such as Kozak sequences which are known to be involved in how the ribosome initiates translation of many genes. 5' UTRs also have been known to form secondary structures which are involved in elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intron sequences) may also be used as regions or subregions which may increase protein production as well as polynucleotide levels.

[0150] Natural or wild type 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the nucleic acid vaccines.

[0151] The UTR from any gene may be incorporated into the regions of the polynucleotides of the nucleic acid vaccines. Alternatively, artificial UTRs, which are not variants of wild type regions, may also be used in the polynucleotides of the nucleic acid vaccines. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered inorientation or location as compared to a reference sequence. As a non-limiting example, a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs from a different parental sequence.

[0152] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5 ’UTR having a sequence of SEQ ID NO. 149 (DNA) or SEQ ID NO. 150 (RNA). In some embodiments, the 5’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO. 149 (DNA) or SEQ ID NO. 150 (RNA).

[0153] In some embodiments, the 5 ’UTR is directly 5’ of the start codon of the coding region of the nucleic acid vaccine. In some embodiments, the 5 ’UTR is directly 5’ of the signal peptide of the nucleic acid vaccine. In some embodiments, the 5 ’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 5’ of the start codon of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO. 149 (DNA) or SEQ ID NO. 150 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO. 149 (DNA) or SEQ ID NO. 150 (RNA).

[0154] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3 ’UTR having a sequence of SEQ ID NO. 151 (DNA) or SEQ ID NO. 152 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO. 151 (DNA) or SEQ ID NO. 152 (RNA). In some embodiments, the 3’UTR is directly 3’ of the last codon of the coding region of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the last codon of the coding region of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO. 151 (DNA) or SEQ ID NO. 152 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO. 151 (DNA) or SEQ ID NO. 152 (RNA).

[0155] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO. 153 (DNA) or SEQ ID NO. 154 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO. 153 (DNA) or SEQ ID NO. 154 (RNA). In some embodiments, the 3’UTR is directly 3’ of the last codon of the coding region of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the last codon of the coding region of the nucleic acidvaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO. 153 (DNA) or SEQ ID NO. 154 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO. 153 (DNA) or SEQ ID NO. 154 (RNA).

[0156] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO. 155 (DNA) or SEQ ID NO. 156 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO. 155 (DNA) or SEQ ID NO. 156 (RNA). In some embodiments, the 3’UTR is directly 3’ of the last codon of the coding region of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the last codon of the coding region of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO. 155 (DNA) or SEQ ID NO. 156 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO. 155 (DNA) or SEQ ID NO. 156 (RNA).

[0157] The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence of SEQ ID NO. 157 (DNA) or SEQ ID NO. 158 (RNA). In some embodiments, the 3’ UTR of the polynucleotides of the nucleic acid vaccines disclosed herein consist of the nucleic acid sequence of SEQ ID NO. 157 (DNA) or SEQ ID NO. 158 (RNA). In some embodiments, the 3’UTR is directly 3’ of the last codon of the coding region of the nucleic acid vaccine. In some embodiments, the 3’UTR is 1, 2, 3, 4, 5, 6 or more nucleotides 3’ of the last codon of the coding region of the nucleic acid vaccine. The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO. 157 (DNA) or SEQ ID NO. 158 (RNA). The polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 3’UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO. 157 (DNA) or SEQ ID NO. 158 (RNA).mRNA Components: Cap Structure

[0158] In some embodiments, the polynucleotides of the nucleic acid vaccines disclosed herein may comprise a 5’ cap structure. The 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsible formRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[0159] In some embodiments, the 5 ’ terminal capping region of the polynucleotide of the nucleic acid vaccine may comprise a single cap or a series of nucleotides forming the cap. The capping region may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some examples, the capping region may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. In some embodiments, the cap is absent.

[0160] In some embodiments, the mRNA comprises a capping sequence which comprises a single cap or a series of nucleotides forming the cap. The capping sequence may be from 1 to 10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the caping sequence is absent.

[0161] In some embodiments, cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs may be used in the nucleic acid vaccines. Cap analogs, which may be chemically (e.g., non-enzymatically) or enzymatically synthesized, differ from natural (e.g., endogenous, wild-type or physiological) 5'-caps in their chemical structure, but they retain cap function.

[0162] Endogenous mRNA molecules may be 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-0-methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0163] Modifications to mRNA may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

[0164] Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0165] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5 '-terminal and / or 5'-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxylgroup of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as an mRNA molecule.

[0166] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e., endogenous, wildtype or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or linked to a nucleic acid molecule.

[0167] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5 '-guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., an mRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g., mRNA).

[0168] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-0-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0169] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5 '-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[0170] mRNA may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and / or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5 'cap structures are those which, among other things, have enhanced binding of cap binding proteins, increased half-life, reduced susceptibility to 5' endonucleases and / or reduced 5 'decapping, as compared to synthetic 5 'cap structures known in the art (or to a wild-type, natural or physiological 5 'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5 '-5 '-triphosphate linkage between the 5 '-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation, and the 5 '-terminal nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a highertranslational -competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5*)ppp(5*)N,pN2p (cap 0), 7mG(5*)ppp(5*)NlmpNp (cap 1), and 7mG(5*)-ppp(5')NlmpN2mp (cap 2).

[0171] In some embodiments, the 5' terminal caps may include endogenous caps or cap analogs.

[0172] In some embodiments, a 5' terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1 -methyl -guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.mRNA Components: Tailing Region

[0173] In some embodiments, the polynucleotide of the nucleic acid vaccine, e.g., the mRNA includes a tailing region. Non-liming examples of a tailing region include a poly-A sequence, a poly-C sequence, and / or a polyA-G quartet.

[0174] In some embodiments, the sequence of the tailing region of the polynucleotide of the nucleic acid vaccine may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). If the tailing region is a poly-A tail, the length may be described in units of or as a function of poly-A binding protein binding.

[0175] During RNA processing, a long chain of adenine nucleotides (poly-A tail) may be added to a polynucleotide such as an mRNA molecule in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 80 to approximately 250 residues long, including approximately 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240 or 250 residues long.

[0176] In some embodiments, the length of a poly-A tail, when present, is greater than 30 nucleotides in length (e.g., at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, and 200 nucleotides). In some embodiments, the poly-A tail region thereof includes from about 30 to about 200 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 200, from 50 to 100, from 100 to 200 nucleotides).

[0177] In some embodiments, the poly-A tail is approximately 100 nucleotides in length (SEQ ID NO. 159 for DNA; SEQ ID NO. 160 for RNA).

[0178] In some embodiments, the poly-A tail is designed relative to the length of the overall polynucleotide or the length of a particular region of the polynucleotide. This design may be based on the length of a coding region, the length of a particular feature or region or based on the length of the ultimate product expressed from the polynucleotides.

[0179] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotide or feature thereof. The poly-A tail may also be designed as a fraction of the polynucleotides to which it belongs. In this context, the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct, a construct region or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides for Poly-A binding protein may enhance expression.

[0180] In some embodiments, spacer regions may be present in the polynucleotide such as, but not limited to, the polyadenylation sequence. There may be one or more such spacer regions present.

[0181] Additionally, multiple distinct mRNA may be linked together to the PABP (Poly-A binding protein) through the 3 '-end using modified nucleotides at the 3 '-terminus of the poly-A tail. Transfection experiments can be conducted in relevant cell lines at, and protein production can be assayed by ELISA at 12 hour, 24 hour, 48 hour, 72 hour and day 7 post-transfection.

[0182] In some embodiments, the mRNA are designed to include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. Signal Sequences

[0183] In some embodiments, the polynucleotides of the nucleic acid vaccines may also encode additional features which may facilitate the trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence, which is also referred to as a signal peptide, which is from about 9 to 200 nucleotides (3-60 amino acids) in length and usually incorporated at the 5' terminus of the coding region or the N-terminus of the encoded polypeptide, respectively. While not wishing to be bound by theory, the addition of these sequences may results in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.

[0184] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO. 88 (DNA) or SEQ ID NO. 89 (RNA). In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a signal sequence comprising SEQ ID NO. 87.

[0185] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO. 88 (DNA) or SEQ ID NO. 89 (RNA). In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a signal sequence comprising SEQ ID NO. 87.Codon Optimization

[0186] The polynucleotides of the nucleic acid vaccines, their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove / add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the mRNA. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the ORF sequence is optimized using optimization algorithms. Examples for codon options for each amino acid include: Alanine (A) - GCT, GCC, GCA, GCG; Cysteine (C) - TGT, TGC; Aspartic acid (D) - GAT, GAC; Glutamic acid (E) - GAA, GAG; Phenylalanine (F) - TTT, TTC; Glycine (G) - GGT, GGC, GGA, GGG; Histidine (H) - CAT, CAC; Isoleucine (I) - ATT, ATC, ATA; Lysine (K) - AAA, AAG; Leucine (L) - CTT, CTC, CTA, CTG, TTA, TTG; Methionine (M) - ATG; Asparagine (N) - AAT, AAC; Proline (P) - CCT, CCC, CCA, CCG; Glutamine (Q) - CAA, CAG; Arginine (R) - CGT, CGC, CGA, CGG, AGA, AGG; Serine (S) - TCT, TCC, TCA, TCG, AGT, AGC; Selenocysteine (Sec) - UGA in mRNA in presence of Selenocysteine insertion element (SECTS); Threonine (T) - ACT, ACC, ACA, ACG; Valine (V) - GTT, GTC, GTA, GTG; Tryptophan (W) - TGG; Tyrosine (Y) - TAT, TAC; Stop codons (stop) -TAA, TAG, TGA.

[0187] In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Nonlimiting examples of vectors include, but are not limited to, plasmids, viruses, cosmids, and artificial chromosomes.RNA Modi fications

[0188] In some aspects, the originator constructs or benchmark constructs may contain one or more modified nucleotides such as, but not limited to, sugar modified nucleotides, nucleobase modifications and / or backbone modifications. In some aspects, the originator constructs or benchmark constructs may contain combined modifications, for example, combined nucleobase and backbone modifications.

[0189] In some embodiments, the modified nucleotide may be a sugar-modified nucleotide. Sugar modified nucleotides include, but are not limited to 2'-fluoro, 2'-amino and 2'-thio modified ribonucleotides, e.g., 2'-fluoro modified ribonucleotides. Modified nucleotides may be modified on thesugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moieties may be, or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.

[0190] In some embodiments, the modified nucleotide may be a nucleobase-modified nucleotide.

[0191] In some embodiments, the modified nucleotide may be a backbone-modified nucleotide. In some embodiments, the originator constructs or benchmark constructs may further comprise other modifications on the backbone. A normal “backbone”, as used herein, refers to the repeating alternating sugar-phosphate sequences in a DNA or RNA molecule. The deoxyribose / ribose sugars are joined at both the 3 '-hydroxyl and 5 '-hydroxyl groups to phosphate groups in ester links, also known as "phosphodiester," bonds / linker (PO linkage). The PO backbones may be modified as “phosphorothioate backbone (PS linkage). In some cases, the natural phosphodiester bonds may be replaced by amide bonds but the four atoms between two sugar units are kept. Such amide modifications can facilitate the solid phase synthesis of oligonucleotides and increase the thermodynamic stability of a duplex formed with siRNA complement.

[0192] Modified bases refer to nucleotide bases such as, but not limited to, adenine, guanine, cytosine, thymine, uracil, xanthine, inosine, and queuosine that have been modified by the replacement or addition of one or more atoms or groups. Some examples of modifications on the nucleobase moieties include, but are not limited to, alkylated, halogenated, thiolated, aminated, amidated, or acetylated bases, individually or in combination. More specific examples include, for example, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N, -dimethyladenine, 2-propyladenine, 2-propylguanine, 2-aminoadenine, 1 -methylinosine, 3 -methyluridine, 5 -methylcytidine, 5 -methyluridine and other nucleotides having a modification at the 5 position, 5-(2-amino)propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1 -methyladenosine, 2-methyladenosine, 3 -methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5 -methyloxyuridine, deazanucleotides such as 7-deaza-adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5-methyl-2 -thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5 -methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides .

[0193] The originator constructs and / or benchmark constructs may include one or more substitutions, insertions and / or additions, deletions, and covalent modifications with respect to reference sequences, in particular, the parent RNA.

[0194] In some embodiments, the originator constructs and / or benchmark constructs includes one or more post-transcriptional modifications (e.g., capping, cleavage, polyadenylation, splicing, poly-A sequence, methylation, acylation, phosphorylation, methylation of lysine and arginine residues, acetylation, and nitrosylation of thiol groups and tyrosine residues, etc). The one or more post-transcriptional modifications can be any post-transcriptional modification, such as any of the more than one hundred different nucleoside modifications that have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197) In some embodiments, the first isolated nucleic acid comprises messenger RNA (mRNA). In some embodiments, the originator constructs and / or benchmark constructs comprise at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5 -aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5 -hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1 -carboxymethyl -pseudouridine, 5 -propynyl -uridine, 1-propynyl -pseudouridine, 5 -taurinomethyluridine, 1 -taurinomethyl -pseudouridine, 5 -taurinomethyl -2 -thio-uridine, 1 -taurinomethyl -4-thio-uridine, 5 -methyl -uridine, 1 -methyl -pseudouridine, 4-thio-l -methyl -pseudouridine, 2-thio-l-methyl -pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2 -thiopseudouridine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5 -aza-cytidine, pseudoisocytidine, 3 -methyl -cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5 -hydroxymethylcytidine, 1 -methyl -pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio- 1 -methyl -pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, 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, and 4-methoxy-1 -methyl -pseudoisocytidine. In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2 -aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In some embodiments, mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1 -methyl -inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl -6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0195] The originator constructs and / or benchmark constructs may include any useful modification, such as to the sugar, the nucleobase, or the intemucleoside linkage (e.g., to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the intemucleoside linkage. Modifications may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.

[0196] In some embodiments, the originator constructs and / or benchmark constructs includes at least one N(6)methyladenosine (m6A) modification to increase translation efficiency. In some embodiments, the N(6)methyladenosine (m6A) modification can reduce immunogenicity of the originator constructs and / or benchmark constructs.

[0197] In some embodiments, the modification may include a chemical or cellular induced modification. For example, some nonlimiting examples of intracellular RNA modifications are described by Lewis and Pan in "RNA modifications and structures cooperate to guide RNA-protein interactions" from Nat Reviews Mol Cell Biol, 2017, 18:202-210.

[0198] In some embodiments, chemical modifications to the RNA may enhance immune evasion. The RNA may be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5' end modifications (phosphorylation (mono-, di- and tri-), conjugation, inverted linkages, etc.), 3' end modifications (conjugation, DNA nucleotides, inverted linkages, etc.), base modifications (e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners), removal of bases (abasic nucleotides), or conjugated bases. The modified ribonucleotide bases may also include 5 -methylcytidine and pseudouridine. In some embodiments, base modifications may modulate expression, immune response, stability, subcellular localization, to name a few functional effects, of the RNA. In some embodiments, the modificationincludes a bi-orthogonal nucleotides, e.g., an unnatural base. See for example, Kimoto et al, Chem Commun (Camb), 2017, 53: 12309, DOI: 10.1039 / c7cc06661a, which is hereby incorporated by reference.

[0199] In some embodiments, sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar one or more RNA may, as well as backbone modifications, include modification or replacement of the phosphodiester linkages. Specific examples of modifications include modified backbones or no natural intemucleoside linkages such as intemucleoside modifications, including modification or replacement of the phosphodiester linkages. RNA having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this application, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their intemucleoside backbone can also be considered to be oligonucleosides. In particular embodiments, the RNA can include ribonucleotides with a phosphorus atom in its intemucleoside backbone.

[0200] Modified RNA backbones may include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates such as 3 '-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates such as 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. In some embodiments, the RNA may be negatively or positively charged.

[0201] The modified nucleotides can be modified on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another intemucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).

[0202] The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNAhave increased nuclease resistance and subsequently a longer half-life in a cellular environment.Phosphorothioate linked to the RNA is expected to reduce the innate immune response through weaker binding / activation of cellular innate immune molecules.

[0203] In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-O-( 1 -thiophosphate)-adenosine, 5'-O-( 1 -thiophosphate)-cytidine (a-thio-cytidine), 5'-O-( 1 -thiophosphate) -guanosine, 5'-O-(l-thiophosphate)-uridine, or 5'-O-(l-thiophosphate)-pseudouridine).

[0204] Other intemucleoside linkages that may be employed according to the present disclosure, including intemucleoside linkages which do not contain a phosphorous atom, are described herein.

[0205] In some embodiments, the RNA may include one or more cytotoxic nucleosides. Cytotoxic nucleoside may include, but are not limited to, adenosine arabinoside, 5 -azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, cladribine, clofarabine, cytarabine, cytosine arabinoside, l-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine, decitabine, 5 -fluorouracil, fludarabine, floxuridine, gemcitabine, a combination of tegafur and uracil, tegafur ((RS)-5 -fluoro- l-(tetrahydrofuran-2-yl)pyrimidine-2,4(lH,3H)-dione), troxacitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), and 6-mercaptopurine. Additional examples include fludarabine phosphate, N4-behenoyl-l-beta-D-arabinofuranosylcytosine, N4-octadecyl- 1 -beta-D-arabinofuranosylcytosine, N4-palmitoyl- 1 -(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-4055 (cytarabine 5 '-elaidic acid ester).

[0206] In some embodiments, the RNA sequence includes or comprises natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine), nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5 -bromouridine, C5 -fluorouridine, C5 -iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5 -methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemically modified bases, biologically modified bases (e.g., methylated bases), intercalated bases, modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In one embodiment, the RNA sequence includes or comprises incorporates pseudouridine (y). In another embodiment, the RNA sequence includes or comprises 5 -methylcytosine (m5C).

[0207] The RNA may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., naturally-occurring nucleotides, purine or pyrimidine, or any one or more or all of A, G, U, C, I, pU) may or may not be uniformly modified in the RNA, or in a given predetermined sequence region thereof. In some embodiments, the RNA includes a pseudouridine. In some embodiments, the RNA includes an inosine, which may aid in the immune systemcharacterizing the RNA as endogenous versus viral RNAs. The incorporation of inosine may also mediate improved RNA stability / reduced degradation.

[0208] In some embodiments, all nucleotides in the RNA (or in a given sequence region thereof) are modified. In some embodiments, the modification may include an m6A, which may augment expression, an inosine, which may attenuate an immune response, pseudouridine, which may increase RNA stability, or translational readthrough (stagger element), an m5C, which may increase stability, and a 2,2,7-trimethylguanosine, which aids subcellular translocation (e.g., nuclear localization).

[0209] Different sugar modifications, nucleotide modifications, and / or intemucleoside linkages (e.g., backbone structures) may exist at various positions in the RNA. One of ordinary skill in the art can appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of the RNA, such that the function of the RNA is not substantially decreased. A modification may also be a noncoding region modification. The RNA may include 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%).SynthesisEnzymatic MethodsIn Vitro Transcription-Enzymatic Synthesis

[0210] cDNA encoding the polynucleotides of the nucleic acid vaccines described herein may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and polymerase variants.

[0211] In some embodiments, the DNA template is removed from the IVT reaction, using a DNase I enzyme. The digested DNA and nucleotides are then removed during oligo dT purification of the mRNA.This purification method is based on affinity of the poly-A tail of the mRNA to the poly-dT column bed. Centrifugation may be used but may not be required to remove the digested DNA and nucleotides. After purification by a reverse phase column (e.g., SDVB) to remove double stranded RNA from the mRNA, ultrafiltration may be utilized, followed by one or more filtration steps. Following purification, residual DNA may be measured to confirm that the DNA has been removed by using PCR for a region of the plasmid outside of the region transcribed into mRNA. In some embodiments, where concentration of the product is desired, diafiltration methods may be used followed by one or more filtration steps to remove any bioburden (e.g., biomolecules, or other biomaterial).

[0212] Any number of RNA polymerases or variants may be used in the synthesis of the polynucleotides of the nucleic acid vaccine described herein. RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence.

[0213] Polynucleotide or nucleic acid synthesis reactions may be carried out by enzymatic methods utilizing polymerases. Polymerases catalyze the creation of phosphodiester bonds between nucleotides in a polynucleotide or nucleic acid chain. Currently known DNA polymerases can be divided into different families based on amino acid sequence comparison and crystal structure analysis. DNA polymerase I (pol I) or A polymerase family, including the Klenow fragments of E. Coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerases, and the T7 RNA and DNA polymerases, is among the best studied of these families. Another large family is DNA polymerase a (pol a) or B polymerase family, including all eukaryotic replicating DNA polymerases and polymerases from phages T4 and RB69. Although they employ similar catalytic mechanism, these families of polymerases differ in substrate specificity, substrate analog -incorporating efficiency, degree and rate for primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity against inhibitors.Solid-Phase Chemical Synthesis

[0214] In some embodiments, polynucleotides of the nucleic acid vaccines described herein may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer-controlled solid-phase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids.

[0215] In some embodiments, automated solid-phase synthesis is used where the chain is synthesized in 3' to 5' direction. The hydroxyl group in the 3' end of a nucleoside is tethered to a solid support via a chemically cleavable or light-cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG and dT), ribonucleosides (A, C, G, and U), or modified nucleosides, are added to the support-bound nucleoside sequentially. At the end of the synthesis, a cleaving agent such as ammonia or ammonium hydroxide is added to remove all the protecting groups and release the polynucleotide chains from the solid support. Light may also be applied to cleave the polynucleotide chain. The product can then be further purified with high pressure liquid chromatography (HPLC) or electrophoresis.Liquid Phase Chemical Synthesis

[0216] The synthesis of polynucleotides of the nucleic acid vaccines described herein by the sequential addition of monomer building blocks may be carried out in a liquid phase. A covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture has to be purified before adding the next monomer building block. The functional group at one terminal of the chain has to be deprotected to be able to react with the next monomer building blocks. A liquid phase synthesis is labor- and timeconsuming and cannot not be automated. Despite the limitations, liquid phase synthesis is still useful in preparing short polynucleotides in a large scale. Because the system is homogenous, it does not require a large excess of reagents and is cost- effective in this respect.Quanti fication and Puri fication

[0217] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein may be quantified in exosomes or when derived from one or more bodily fluid.

[0218] In the exosome quantification method, a sample of not more than 2 mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof. In the analysis, the level or concentration of a polynucleotide may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker. The assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or combinations thereof.

[0219] These methods afford the investigator the ability to monitor, in real time, the level of polynucleotides remaining or delivered. This is possible because the polynucleotides described herein differ from the endogenous forms due to the structural modifications.

[0220] In some embodiments, the polynucleotide may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified polynucleotide may be analyzed in order to determine if the polynucleotide may be of proper size, check that no degradation of the polynucleotide has occurred. Degradation of the polynucleotide may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC- HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).

[0221] Purification of the polynucleotides of the nucleic acid vaccines described herein may include, but is not limited to, polynucleotide clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGEN- COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EX- IQON® Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The polynucleotides of the nucleic acid vaccines may undergo tangential flow filtration (TFF) to remove process-related impurities and concentrates the mRNA to a target concentration in water for injection. The final nucleic acid vaccine may be filtered to reduce bioburden, and the goal is to have a clear, colorless, aqueous solution with a pH between 5.7 and 8.4. The term “purified” when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant. As used herein, a “contaminant” is any substance which makes another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

[0222] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.II, PHARMACEUTICAL COMPOSITIONS AND ROUTE OF ADMINISTRATION Pharmaceutical Composition and Formulations

[0223] The lipid nanoparticles comprising the nucleic acid vaccines can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection or transduction; (3) permit thesustained or delayed expression of the cargo / payload; (4) alter the biodistribution (e.g., target the viral particle to specific tissues or cell types); (5) increase the translation of encoded protein; (6) alter the release profile of encoded protein; and / or (7) allow for regulatable expression of the cargo / payload.

[0224] Formulations can include, in addition to the lipid nanoparticles of the present disclosure, without limitation, saline, liposomes, other lipid nanoparticles, polymers, peptides, proteins, cells transfected with viral vectors (e.g., for transfer or transplantation into a subject) and combinations thereof.

[0225] Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. As used herein the term “pharmaceutical composition” refers to compositions comprising at least one active ingredient and optionally one or more pharmaceutically acceptable excipients.

[0226] In general, such preparatory methods include the step of associating the active ingredient with an excipient and / or one or more other accessory ingredients. As used herein, the phrase “active ingredient” generally refers either to an originator construct or benchmark construct with a payload region or payload as described herein. In some embodiments, the nucleic acid vaccines and formulations thereof are considered the active ingredient.

[0227] Formulations of the lipid nanoparticles comprising payloads and pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi -dose unit.

[0228] A pharmaceutical composition in accordance with the disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses.

[0229] In some embodiments, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use for humans and for veterinary use. In some embodiments, an excipient may be approved by United States Food and Drug Administration. In some embodiments, an excipient may be of pharmaceutical grade. In some embodiments, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0230] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w / w) of the active ingredient. By way of example, thecomposition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, or at least 80% (w / w) active ingredient.Lipids Nanoparticles (LNP)

[0231] In general, lipid nanoparticles (LNP) can be characterized as small solid or semi-solid particles possessing an exterior lipid layer with a hydrophilic exterior surface that is exposed to the nonlipid nanoparticle environment, an interior space which may aqueous (vesicle like) or non-aqueous (micelle like), and at least one hydrophobic inter-membrane space. Lipid nanoparticle membranes may be lamellar or non -lamellar and may be comprised of 1, 2, 3, 4, 5 or more layers. In some embodiments, lipid nanoparticles may comprise a cargo or a payload into their interior space, into the inter membrane space, onto their exterior surface, or any combination thereof.

[0232] “Lipid particle” or “lipid nanoparticle (i.e., “LNP”) means a lipid formulation that can be used to deliver a cargo, such as a therapeutic nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, and the like). In preferred embodiments, the lipid particle can be used to encapsulate a nucleic acid. In preferred embodiments, the lipid nanoparticle can be formed from an ionizable lipid, a neutral lipid (e.g., a phospholipid), a polymer-conjugated lipid that can prevent aggregation of the nanoparticle (e.g., a PEG-lipid), and optionally a helper lipid (e.g., cholesterol). In some embodiments, a therapeutic nucleic acid (e.g., mRNA) may be encapsulated in the lipid portion of the nanoparticle, thereby protecting it from enzymatic degradation. In another preferred embodiment, the lipid nanoparticle can comprise another component such as a hydrophobic component to improve LNP internalization, immune activation and / or antibody production.

[0233] The present disclosure also provides lipid nanoparticle comprising a nucleic acid vaccine such as a mRNA vaccine.

[0234] In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid of the present disclosure, such as the ionizable lipid of general Formula (I), or more particularly, the compounds in Table 4. Not willing to be bound to any theory, the diester bonds of the lipids provide biodegradability and biocompatibility, such ester bonds are stable at physiological pH, but can be enzymatically hydrolyzed within tissues and cells. The length of the Rl, R2, R3 and R4 groups in Formula (I) can also be adjusted to reach the desired zeta potential, particle size or membrane rigidity.

[0235] In some embodiments, the lipid nanoparticle may comprise any lipid described in the disclosure. In some embodiments, the lipid may be any cationic lipid described in the disclosure. In some embodiments, the lipid nanoparticle may comprise neutral lipids. In some embodiments, the neutral lipid may be a phospholipid, or a derivative thereof. In some embodiments, the lipid may be any phospholipid described in the disclosure. In some embodiments, the lipid may be any cholesterol derivative described in the disclosure. In some embodiments, a polymer (e.g., PEG) may be conjugated to at least one lipid. Insome embodiments, a PEG-lipid may be used in the lipid nanoparticle and can be any PEG-lipid conjugate described in the disclosure.

[0236] In some embodiments, the lipid nanoparticles can be characterized by their shape. In some embodiments, the lipid nanoparticles are essentially spherical. In some embodiments, the lipid nanoparticles are essentially rod-shaped (i.e., cylindrical). In some embodiments, the lipid nanoparticles are essentially disk shaped.

[0237] In some embodiments, the term “nanoparticle” or “lipid nanoparticle” as used herein refers to any particle ranging in size from 10-1000 nm. The lipid nanoparticle may be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500, 505, 510, 515, 520, 525, 530, 535, 540, 545, 550, 555, 560, 565, 570, 575, 580, 585, 590, 595, 600, 605, 610, 615, 620, 625, 630, 635, 640, 645, 650, 655, 660, 665, 670, 675, 680, 685, 690, 695, 700, 705, 710, 715, 720, 725, 730, 735, 740, 745, 750, 755, 760, 765, 770, 775, 780, 785, 790, 795, 800, 805, 810, 815, 820, 825, 830, 835, 840, 845, 850, 855, 860, 865, 870, 875, 880, 885, 890, 895, 900, 905, 910, 915, 920, 925, 930, 935, 940, 945, 950, 955, 960, 965, 970, 975, 980, 985, 990, 995, or 1000 nm.

[0238] Lipid nanoparticles typically can have a particle size, e.g., expressed as a mean diameter, ranging from 30 nm to 200 nm, from 40 nm to 180 nm, from 50 nm to 150 nm, from 60 nm to 130 nm, from 60 nm to 120 nm, from 60 nm to 110 nm, from 60 nm to 100 nm, from 60 nm to 90 nm, from 70 nm to 110 nm, from 70 nm to 100 nm, from 80 nm to 100 nm, from 90 nm to 100 nm, from 70 to 90 nm, from 80 nm to 90 nm, or from 70 nm to 80 nm. In some embodiments, the particle size can be about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm and are substantially non-toxic. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, are resistant in aqueous solution to degradation with a nuclease. In some embodiments, the particle size of the lipid nanoparticle is approximately 50-100 nm.

[0239] In some embodiments, a population of lipid nanoparticles, such as those resulting from the same formulation, may be characterized by measuring the uniformity of size, shape, or mass of the particles in the population, uniformity may be expressed in some embodiments as the polydispersity index (PI) of the population. In some embodiments uniformity may be expressed in some embodiments as the disparity (D) of the population. The terms “polydispersity index” and “disparity” are understood herein tobe equivalent and may be used interchangeably. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 and 1. In some embodiments, a population of lipid nanoparticles resulting from a giving formulation can have a PI of less than about 1, less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1. In some embodiments, a population of lipid nanoparticles resulting from a given formulation can have a PI of between about 0.1 to 1, 0.1 to 0.8, 0.1 to 0.6, 0.1 to 0.4, 0.1 to 0.2, 0.2 to 1, 0.2 to 0.8, 0.2 to 0.6, 0.2 to 0.4, 0.4 to 1, 0.4 to 0.8, 0.4 to 0.6, 0.6 to 1, 0.6 to 0.8, and 0.8 to 1. In some embodiments, the lipid nanoparticle may have PI ranging between about 0.01 to 0.3, 0.02-0.3, 0.03-0.3, 0.04-0.3, 0.05-0.3, 0.06-0.3, 0.07-0.3, 0.08-0.3, 0.09-0.3, 0.1-0.3, 0.11-0.3, 0.12-0.3, 0.13-0.3, 0.14-0.3, 0.15-0.3, 0.16-0.3, 0.17-0.3, 0.18-0.3, 0.19-0.3, 0.2-0.3, 0.21-0.3, 0.22-0.3, 0.23-0.3, 0.24-0.3, 0.25-0.3, 0.26-0.3, 0.27-0.3, 0.28-0.3, 0.29-0.3, 0.01 to 0.25, 0.02-0.25, 0.03-0.25, 0.04-0.25, 0.05-0.25, 0.06-0.25, 0.07-0.25, 0.08-0.25, 0.09-0.25, 0.1-0.25, 0.11-0.25, 0.12-0.25, 0.13-0.25, 0.14-0.25, 0.15-0.25, 0.16-0.25, 0.17-0.25, 0.18-0.25, 0.19-0.25, 0.2-0.25, 0.21-0.25, 0.22-0.25, 0.23-0.25, 0.24-0.25, 0.01 to 0.2, 0.02-0.2, 0.03-0.2, 0.04-0.2, 0.05-0.2, 0.06-0.2, 0.07-0.2, 0.08-0.2, 0.09-0.2, 0.1-0.2, 0.11-0.2, 0.12-0.2, 0.13-0.2, 0.14-0.2, 0.15-0.2, 0.16-0.2, 0.17-0.2, 0.18-0.2, 0.19-0.2, 0.01 to 0.15, 0.02-0.15, 0.03-0.15, 0.04-0.15, 0.05-0.15, 0.06-0.15, 0.07-0.15, 0.08-0.15, 0.09-0.15, 0.1-0.15, 0.11-0.15, 0.12-0.15, 0.13-0.15, 0.14-0.15, 0.01 to 0.1, 0.02-0.1, 0.03-0.1, 0.04-0.1, 0.05-0.1, 0.06-0.1, 0.07-0.1, 0.08-0.1, 0.09-0.1, 0.01 to 0.05, 0.02-0.0.5, 0.03-0.0.5, or 0.04-0.05.

[0240] In some embodiments, the total mole percentage of the lipid(s) in the LNP is between about 10% to about 95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31 % to about 40%, between about 41 % to about 50%, between about 51 % to about 60%, between about 61% to about 70%, between about 71% to about 80%, between about 81% to about 90%, or between about 91% to about 95%.

[0241] In some embodiments, the lipid nanoparticles of the present disclosure can be used for lyophilized vaccines. Lyophilization of vaccine containing lipid nanoparticles, through freeze-dried techniques, would allow easier distribution and storage of the pharmaceutical compositions. The rehydration of the freeze-dried formulation can be completed by the user right before injection.Types of Lipids and Possible Components of LNPs

[0242] Lipid nanoparticles generally comprise cholesterol (aids in stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and also may aid in endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and “shields” the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (complexes negatively charged RNA and enhances endosomal escape), which form the LNP -forming composition.

[0243] “Lipid” means an organic compound that comprises an ester of fatty acid and is characterized by being insoluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.Ionizable Lipids

[0244] Provided herein to be used with the nucleic acid vaccines are ionizable lipids, more particularly ionizable diester lipids. The ionizable lipids may be cationic lipids.

[0245] In some embodiments, ionizable lipid compounds of the present disclosure comprise at least two ester bonds (-CO-O- or -O-CO-). Ester bonds present the particularity of being biodegradable. In some embodiments, compounds of the present disclosure can further comprise one secondary amino group. In some embodiments, compounds of the present disclosure further comprise at least one terminal amino group, wherein the amino group may be substituted with at least one lower alkyl group (e.g., Cl-C3 alky groups), which may be further substituted. In some embodiments, the terminal amino group can be NH2, a primary amino group, a secondary amino group, or a tertiary amino group. In some embodiments, the terminal amino group can be N(CH3)2, -bXCFLXCFECIL), dS^CFLXCFECFEOH), -N CEECILOHX, or N((CH2)2O(CO)CH3)2, to name a few examples. The ionizable lipids of the present disclosure can be characterized in that the two ester bonds are separated by two tertiary carbon atoms, a first one of the two tertiary carbon atoms being substituted with an alkyl chain and the second one of the two tertiary carbon atoms being substituted with a hydrocarbon chain bearing the terminal amino group. The hydrocarbon chain bearing the terminal amino group optionally comprises a nitrogen atom within the chain, this nitrogen atom being itself optionally substituted. These ionizable lipids may be obtained at high purity, and lipid nanoparticles made therefrom can present high stability.

[0246] In some embodiments, the ionizable lipid compound of the present disclosure can have a structure of Formula (I):pharmaceutically acceptable salt thereof, whereinRi and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-;n is a number from 0 to 10;p is a number from 0 to 2;Rs and Re are independently H, or an optionally substituted linear C1-C4 alkyl group; R7 is a linear or branched C1-C6 alkyl;wherein when any of Ri, R2, Rs and R4 represents an alkenyl group, the alkenyl group independently comprises from one to six C=C bonds each independently having the E or Z configuration; wherein when any of Ri, R2, Rs and R4 represents an alkynyl group, the alkynyl group independently comprises from one to six C=C bonds.

[0247] In some embodiments, in the structure of Formula (I), m can be an integer from 1 to 12, or m can be an integer from 1 to 11, or from 1 to 10, or from 1 to 9, or from 1 to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1 to 3, or m can be 1 or 2. In some embodiments, in the structure of Formula (I), m can be 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12.

[0248] In some embodiments, in the structure of Formula (I), n can be an integer from 0 to 10, or n can be an integer from 0 to 9, or from 0 to 8, or from 0 to 7, or from 0 to 6, or from 0 to 5, or from 0 to 4, or from 0 to 3, or from 0 to 2, or n can be 0, 1 or 2. In some embodiments, in the structure of Formula (I), n can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10.

[0249] In some embodiments, in the structure of Formula (I), p can be an integer from 0 to 2, or p can be 0 or 1, or p can be 1 or 2, or p can be 0, or 1, or 2.

[0250] In some embodiments, in the structure of Formula (I), X is -CH2-.

[0251] In some embodiments, in the structure of Formula (I), X is -NH-.

[0252] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is a linear or branched C1-C6 alkyl.

[0253] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is a linear or branched C1-C4 alkyl.

[0254] In some embodiments, in the structure of Formula (I), X is -NR7- and R7 is C1-C2 alkyl.

[0255] In some embodiments, in the structure of Formula (I), X is -NMe-.

[0256] In some embodiments, when any of the alkyl, alkenyl and / or alkynyl groups in the substituents of the Formula (I) is substituted, these groups can independently be substituted with one or more halogen, hydroxyl, acetoxy, alkoxycarbonyl, formyl, acyl, thiocarbonyl, alkoxyl, phosphoryl, phosphate, phosphonate, a phosphinate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamido, sulfonyl, heterocyclyl, aralkyl, an aromatic moiety or an heteroaromatic moiety.

[0257] In some embodiments, when any the alkyl, alkenyl and / or alkynyl groups in the substituents of the Formula (I) are substituted, these groups can independently be substituted with one or more halogen, hydroxyl, acetoxy, Cl-C4alkoxy carbonyl, formyl, Cl-C4acyl, Cl-C4alkoxyl, amino, -(CO)NHC1-C4alkyl, -NH(CO)C1-C4alkyl, amidine, (Cl-C4alkyl)2C=N-, cyano, nitro, azido, sulfhydryl, Cl-C4alkylthio, sulfamoyl, -(SO2)NHC1-C4alkyl, -NH(SO2)C1-C4alkyl, -(SO2)Cl-C4alkyl, 3- to 6-membered heterocyclyl, 6- to 10-membered aralkyl, or 5- to 10-membered aromatic or heteroaromatic moiety.

[0258] In some embodiments, when any alkyl, alkenyl and / or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.

[0259] In some embodiments, when any alkyl, alkenyl and / or alkynyl group groups in the substituents of the Formula (I) are substituted, these groups can be independently substituted with one or more hydroxyl or acetoxy.

[0260] In some embodiments, the ionizable diester lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) whereinRi and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-;n is a number from 0 to 8;p is 0 or 1;Rs and R., are independently an optionally substituted C1-C4 alkyl group;R7 is a linear or branched C1-C4 alkyl.

[0261] In some embodiments, the ionizable diester lipid, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) whereinRi and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;m is a number from 1 to 12;X is -CH2-, -NH- or -NR7-;n is a number from 0 to 8;p is 0 or 1;Rs and R., are independently an optionally substituted C1-C4 alkyl group; andR7 is a linear or branched C1-C4 alkyl;wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, Cl-C4alkoxycarbonyl, formyl, Cl-C4acyl, Cl-C4alkoxyl, amino, -(CO)NHC1-C4alkyl, -NH(CO)C1-C4alkyl, amidine, (Cl-C4alkyl)2C=N-, cyano, nitro, azido, sulfhydryl, Cl-C4alkylthio, sulfamoyl, -(SO2)NHC1-C4alkyl, -NH(SC>2)C1-C4alkyl, -(SO2-)C1-C4alkyl, 3- to 6-membered heterocyclyl, 6- to 10-membered aralkyl, or 5- to 10-membered aromatic or heteroaromatic moiety.

[0262] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:Ri and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;m is a number from 1 to 8;X is -CH2-, -NH- or -NR7-;n is a number from 0 to 8;p is 0 or 1;Rs and R., are independently an optionally substituted C1-C4 alkyl group;R7 is a C1-C2 alkyl.

[0263] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:Ri and R4 are independently an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;R2 and R3 are independently H or an optionally substituted linear or branched C8-C20 alkyl, an optionally substituted linear or branched C8-C20 alkenyl, or an optionally substituted linear or branched C8-C20 alkynyl group;m is a number from 1 to 8;X is -CH2-, -NH- or -NR7-;n is a number from 0 to 8;p is 0 or 1;Rs and R., are independently an optionally substituted C1-C4 alkyl group;R7 is a C1-C2 alkyl;wherein when any alkyl, alkenyl and / or alkynyl group is substituted, this group is independently substituted with one or more halogen, hydroxyl, acetoxy, amino, cyano, nitro, azido, or sulfhydryl.

[0264] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:Ri and R4 are independently linear or branched C8-C20 alkyl, linear or branched C8-C20 alkenyl, or linear or branched C8-C20 alkynyl group;R2 and R3 are independently H, linear or branched C8-C20 alkyl, linear or branched C8-C20 alkenyl, or linear or branched C8-C20 alkynyl group;m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-;n is a number from 0 to 8;p is 0 or 1;Rs and R., are independently a C1-C4 alkyl group optionally substituted with hydroxyl or acetoxy.

[0265] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:Ri and R4 are independently linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8-C20 alkynyl group;R2 and R3 are independently H, linear C8-C20 alkyl, linear C8-C20 alkenyl, or linear C8-C20 alkynyl group;m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-;n is a number from 0 to 8;p is a number from 0 to 1 ; andRs and Re are independently a C1-C4 alkyl group.

[0266] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of Ri, R2, Rs and R4 is C8-C20 alkenyl, the alkenyl group comprises one to four C=C bonds, and when any of Ri, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two C^C bonds.

[0267] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of Ri, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one to three C=C bonds, and when any of Ri, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two t ’=C bonds.

[0268] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of Ri, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one or two C=C bonds, and when any of Ri, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one or two C^C bonds.

[0269] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein when any of Ri, R2, R3 and R4 is C8-C20 alkenyl, the alkenyl group comprises one C=C bonds, and when any of Ri, R2, R3 and R4 is C8-C20 alkynyl, the alkynyl group comprises one C^C bonds.

[0270] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one to four C=C bonds.

[0271] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one to three C=C bonds.

[0272] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one or two C=C bonds.

[0273] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri, R2, R3 and R4 independently represent C8-C20 alkenyl, the alkenyl group comprising one C=C bonds.

[0274] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein:Ri and R4 are independently linear C8-C20 alkyl or linear C8-C20 alkenyl;R2 and R3 are independently H or linear C8-C20 alkyl or linear C8-C20 alkenyl; m is a number from 1 to 8;X is -CH2-, -NH- or -NMe-;n is a number from 0 to 8;p is a number from 0 to 1 ; andRs and Rs are independently C1-C4 alkyl.

[0275] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical.

[0276] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R2 and Rs are H or linear C8-C18 alkyl, preferably H or C10-C16 alkyl, more preferably H or C14 alkyl.

[0277] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R2 and Rs are H.

[0278] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical and represent a linear C8-C18 alkyl or linear C8-C18 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

[0279] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one to four C=C bonds.

[0280] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one to three C=C bonds.

[0281] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one or two C=C bonds.

[0282] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Ri and R4 are identical and represent a linear C10-C14 alkyl or linear C6-C16 alkenyl, wherein the alkenyl groups comprise one C=C bonds.

[0283] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein R5 and R; are independently a C1-C2 alkyl group.

[0284] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formula (I) wherein Rs and Re are identical.

[0285] In some embodiments, the ionizable diester lipid compound can have a structure of Formulaor a pharmaceutically acceptable salt thereof,wherein m is a number from 1 to 12; n is a number from 0 to 8; p is 0 or 1; q is a number from 0 to 8; r is a number from 1 to 15; s in number from 0 to 5; t is a number from 0 to 6; X is -CH2-, -NH- or -NMe-; and LI and L2 are independently a number from 0 to 3, andwherein the C=C double bonds present in any of the Formulas (II), (12), (13), (14), (15), (16), (17), or (18) independently have the E or Z configuration.

[0286] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15) or (16), q can be a number from 0 to 11, from 0 to 10, from 0 to 9, from 0 to 8, from 0 to 7, from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or q can be 0 or 1, or q can be 1 or 2, or q can be 0, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11.

[0287] In some embodiments, in the structure of Formulas (17), s can be a number from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or s can be 0 or 1, or s can be 1 or 2, or s can be 0, or 1, or 2, or 3, or 4, or 5.

[0288] In some embodiments, in the structure of Formulas (18), t can be a number from 0 to 6, from 0 to 5, from 0 to 4, from 0 to 3, from 0 to 2, or t can be 0 or 1, or t can be 1 or 2, or t can be 2 or 3, or t can be 3 or 4, or 1, or 2, or 3, or 4, or 5, or 6.

[0289] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -CH2-.

[0290] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -NH-.

[0291] In some embodiments, in the structures of Formulas (II), (12), (13), (14), (15), (16), (17), or (18), X is independently -NMe-.

[0292] In some embodiments, the ionizable lipid compound, or the pharmaceutically acceptable salt thereof, can have a structure of Formulas (13), (14), (15), (16), (17), or (18) wherein X is -CH2-.

[0293] In some embodiments, the ionizable diester lipid compound can have a structure of Formula1 to 8; and q is a number from 0 to 11.

[0294] In some embodiments, the ionizable diester lipid compound can have a structure of Formulaor a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0295] In some embodiments, the ionizable diester lipid compound can have a structure of Formulaor a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0296] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I2a):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 7.

[0297] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I2b):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 7.

[0298] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (I3a):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0299] In some embodiments, the ionizable diester lipid compound can have a structure of Formula1 to 8; q is a number from 0 to 11; and r is a number from 1 to 13.

[0300] In some embodiments, the ionizable diester lipid compound can have a structure of Formulaor a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 11.

[0301] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (16a):or a pharmaceutically acceptable salt thereof, and wherein m is a number from 3 to 6; n is a number from 1 to 8; and q is a number from 0 to 8.

[0302] In some embodiments, the ionizable diester lipid compound can have a structure of Formulaor a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and s is a number from 0 to 5.

[0303] In some embodiments, the ionizable diester lipid compound can have a structure of Formula (18a):or a pharmaceutically acceptable salt thereof, wherein m is a number from 3 to 6; n is a number from 1 to 8; and t is a number from 0 to 6.

[0304] Each of the C=C double bonds present in the Formulas (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (He), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a), or (I8a) and / or in the compounds represented in Table 4, can independently have the E or Z configuration.

[0305] In some embodiments, the compounds of Formulas (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a), or (I8a) and / or in the compounds represented in Table 4 can be in the form of any enantiomers, diastereoisomers, cis or trans geometric isomers, or mixtures thereof.

[0306] In some embodiments, the ionizable diester lipid compounds of the present disclosure, without being limited to, can be selected from the group consisting of Compounds 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 213, 214, 215, 216, 217 and 218 of Table 4, or a pharmaceutically acceptable salt thereof.

[0307] In some embodiments, the ionizable diester lipid compounds can be in the form of any enantiomer and / or any diastereoisomer thereof, o r any mixture thereof.Table 4: Non-Limiting Examples of o nizable Lipid Compounds

[0308] In some embodiments, the ionizable lipid compounds is an ionizable disulfide lipid compound in the form of any enantiomer and / or any diastereoisomer thereof, or any mixture thereof. In some embodiments, the ionizable lipid compounds of the present disclosure, without being limited to, can be Compound 109 or a pharmaceutically acceptable salt thereof.

[0309] The cis (Z) or trans (E) stereochemistry as drawn in the chemical structures of Table 4 or Compound 109 was randomly attributed. In other words, each of the C=C bonds in the chemical structures of Table 4 or Compound 109 can independently have the cis or trans configuration. In some embodiments, the compounds of Table 4 or Compound 109 can be in the form of mixtures of cis or trans geometric isomers.

[0310] In some embodiments, the ionizable diester lipid compounds of Table 4 or Compound 109 can be in the form of any enantiomers, any diastereoisomers, any cis or trans geometric isomers, or any mixtures thereof.

[0311] The term “compound”, as used herein, is meant to embrace all stereoisomers, geometric isomers, tautomers, and isotopes of a depicted or described structure associated with thecompound. When referring to compound features or substituents, the terms “optional” or “optionally” refer to a feature or substituent that may or may not occur. For example,“optionally substituted alkyl” encompasses both “alkyl” and “substituted alkyl” as defined below. It will be understood by those skilled in the art, with respect to any group containing one or more substituents,that such groups are not intended to introduce any substitution or substitution patterns that are sterically impractical, synthetically non-feasible and / or inherently unstable.

[0312] The compounds herein described may have asymmetric centers, geometric centers (e.g., double bond), or both. All chiral, diastereomeric, racemic forms and all geometric isomeric forms of a structure are intended, unless the specific stereochemistry or isomeric form is specificallyindicated. Compounds of the present disclosure containing an asymmetrically substituted atom may be isolated in optically active or racemic forms. It is well known in the art how to prepare optically active forms, such as by resolution of racemic forms, by synthesis from optically active starting materials, or through use of chiral auxiliaries. Geometric isomers of olefins, C=N double bonds, or other types of double bonds may be present in the compounds described herein, and all such stable isomers are included in the present disclosure. Specifically, cis and trans geometric isomers of the compounds of the present disclosure may also exist and may be isolated as a mixture of isomers or as separated isomeric forms.

[0313] Compounds described herein also embrace tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

[0314] Compounds described herein also embrace all the isotopes of the atoms occurring in the intermediate or final compounds. “Isotopes” refers to atoms having the same atomic number but different mass numbers resulting from a different number of neutrons in the nuclei. Thus, by way of example, each individual hydrogen atom present in formula (200) may be present as a ’H,2H (deuterium) or3H (tritium) atom, preferably ’H or2H. Similarly, by way of example, each individual carbon atom present in formula (200) may be present as a12C,13C or14C atom, preferably12C.

[0315] The compounds or structures and salts of the present disclosure can be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.Neutral Lipids

[0316] In some embodiments, the lipid nanoparticle can also include at least one neutral lipid. In some embodiments, the neutral lipids may be phospholipids, or derivatives thereof. Examples of phospholipids suitable for use in the present disclosure include, but are not limited to: dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine(DOPC), neutral DOPC (l,2-dioleoyl-sn-glycero-3-phosphocholine), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), l,2-distearoyl-sn-glycero-3 -phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), l-myristoyl-2 -palmitoyl phosphatidylcholine (MPPC), 1-palmitoyl -2 -myristoyl phosphatidylcholine (PMPC), 1 -palmitoyl -2-stearoyl phosphatidylcholine (PSPC), l,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1 -stearoyl -2 -palmitoyl phosphatidylcholine (SPPC), l,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphophatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof.

[0317] In some embodiments, the preferred phospholipids are distearoylphosphatidylcholine (DSPC) and dioleoylphosphatidylethanolamine (DOPE).

[0318] In other embodiments, the preferred phospholipids are DSPC, DOPC, DMPC and PE.Helper Lipids

[0319] In some embodiments, the lipid nanoparticle can also include at least one helper lipid. “Helper lipids” are lipids that enhance transfection, such as transfection of the lipid nanoparticle including the payloads and cargos. The mechanism by which the helper lipid enhances transfection may include enhancing particle stability and / or enhancing membrane fusogenicity. Helper lipids include steroids and alkyl resorcinols. Helper lipids suitable for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, cholesterol hemisuccinate, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof.

[0320] In some embodiments, the preferred helper lipid is cholesterol.Polymer-Conjugated Lipids

[0321] In some embodiments, the lipid nanoparticle can further include at least one polymer-conjugated lipid.

[0322] In some embodiments, the polymer-conjugated lipid comprises a polymer conjugated to at least one lipid. In some embodiments, the polymer-conjugated lipid can comprise at least one component that reduces aggregation of particles, at least one component that decreases clearing of the lipid nanoparticle from circulation in a subject, at least component that increases the lipid nanoparticle’s ability to traverse mucus layers, at least one component that decreases a subjects immune response to administration of the lipid nanoparticle, at least one component that modifies membrane fluidity of the lipid nanoparticle, at least one component that contributes to the stability of the lipid nanoparticle, or any combination thereof. In some embodiments, the lipid nanoparticle may be essentially devoid of polymer-conjugated lipid. In some embodiments, the lipid nanoparticle may contain no amount of polymer-conjugated lipid.

[0323] In some embodiments, the polymer present in the polymer-conjugated lipid may comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0324] In some embodiments, the lipid conjugated to the polymer may be selected from, but is not limited to, at least one of the ionizable, neutral, or helper lipids listed previously.

[0325] In some embodiments, the polymer conjugated to at least one lipid is PEG and the polymer-conjugated lipid can be referred to as “PEG-lipid”. In some embodiments, the at least one PEG-lipid may be selected from, but is not limited to at least one of Siglec-IL-PEG-DSPE, R)-2,3-bis(octadecyloxy)propyl- 1 -(methoxypoly(ethyleneglycol)2000)propylcarbamate, PEG-S-DSG, PEG-S-DMG, PEG-PE, PEG-PAA, PEG-OH DSPE C18, PEG-DSPE, PEG-DSG, PEG-DPG, PEG-DOMG, PEG-DMPE Na, PEG-DMPE, PEG-DMG2000, PEG-DMG C14, PEG-DMG 2000, PEG-DMG, PEG-DMA, PEG-Ceramide Cl 6, PEG-C-DOMG, PEG-c-DMOG, PEG-c-DMA, PEG-cDMA, PEGA, PEG750-C-DMA, PEG400, PEG2k-DMG, PEG2k-Cll, PEG2000-PE, PEG2000P, PEG2000-DSPE, PEG2000-DOMG, PEG2000-DMG, PEG2000-C-DMA, PEG2000, PEG200, PEG(2k)-DMG, PEG DSPE C18, PEG DMPE C14, PEG DLPE C12, PEG Click DMG C14, PEG Click C12, PEG Click CIO, N(Carbonyl-methoxypolyethylenglycol-2000)-l,2-distearoyl-sn-glycero3-phosphoethanolamine, Myij52, mPEG-PLA, MPEG-DSPE, mPEG3000-DMPE, MPEG-2000-DSPE, MPEG2000-DSPE, mPEG2000-DPPE, mPEG2000-DMPE, mPEG2000-DMG, mDPPE-PEG2000, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, HPEG-2K-LIPD, Folate PEG-DSPE, DSPE-PEGMA 500, DSPE-PEGMA, DSPE-PEG6000, DSPE-PEG5000, DSPE-PEG2K-NAG, DSPE-PEG2k, DSPE-PEG2000maleimide, DSPE-PEG2000, DSPE-PEG, DSG-PEGMA, DSG-PEG5000, DPPE-PEG-2K, DPPE-PEG, DPPE-mPEG2000, DPPE-mPEG, DPG-PEGMA, DOPE-PEG2000, DMPE-PEGMA, DMPE-PEG2000, DMPE-Peg, DMPE-mPEG2000, DMG-PEGMA, DMG-PEG2000, DMG-PEG, distearoyl-glycerol-polyethyleneglycol, C18PEG750, CI8PEG5000, CI8PEG3000, CI8PEG2000, CI6PEG2000, CI4PEG2000, C18-PEG5000, C18PEG, C16PEG, C16 mPEG (polyethylene glycol) 2000 Ceramide, C14-PEG-DSPE200, C14-PEG2000, C14PEG2000, C14-PEG 2000, C14-PEG, C14PEG, 14:0-PEG2KPE, l,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG2000, (R)-2,3-bis(octadecyloxy)propyl- 1 -(methoxypoly(ethyleneglycol)2000)propylcarbamate, (PEG)-C-DOMG, PEG-C-DMA, and DSPE-PEG-X.

[0326] In some embodiments, the preferred polymer-conjugated lipids are polyethylene glycol (PEG), polypropylene glycol (PPG), poly(2-oxazoline) (POZ), polyamide (ATTA), cationic polymer,polysarcosine (Psar), polyglutamic acid (PGA) and l,2-dimyristoyl-rac-glycero-3 -methoxypolyethylene glycol (PEG-DMG).

[0327] In some embodiments, the preferred polymer-conjugated lipids are PEG-lipids selected from PEG-DMG or PEG-DSG.

[0328] In some embodiments, the preferred PEG-lipid is PEG2k-DMG.

[0329] In some embodiments, the average molecular weight of the polymer moiety (e.g., PEG) of the polymer-conjugated lipid may be between 500 and 20,000 daltons. In some embodiments, the molecular weight of the polymer may be about 500, 1,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 5.500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12.500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500000, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500 19,000, 19,500, or about 20,000 daltons.Hydrophobic Components

[0330] In some embodiments, the lipid nanoparticles can also include another type of lipids, referred to as “hydrophobic components” in the present disclosure. The hydrophobic component is defined as a component, which, during the lipid nanoparticle formation, is incorporated in the lipid bilayer due to its hydrophobic nature. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A and derivatives thereof, P-carotene, withaferin A and a-tocopherol. In some embodiments the hydrophobic component may be selected from the group consisting of cardiolipin, squalene, vitamin A, retinol, P-carotene, withaferin A and a-tocopherol.Excipients

[0331] In some embodiments, pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, anti-oxidants, osmolality adjusting agents, pH adjusting agents and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0332] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents.

[0333] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0334] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, crosslinked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof.

[0335] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrex, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, wax, and lecithin), colloidal clays (e.g. bentonite (aluminum silicate) and VEEGUM® (magnesium aluminum silicate)), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate (TWEEN®20), polyoxyethylene sorbitan (TWEEN®60), polyoxyethylene sorbitan monooleate (TWEEN®80), sorbitan monopalmitate (SPAN®40), sorbitan monostearate (SPAN®60), sorbitan tristearate (SPAN®65), glyceryl monooleate, sorbitan monooleate (SPAN®80)), polyoxyethylene esters (e.g. polyoxyethylene monostearate (MYRJ®45), polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g.CREMOPHOR®), polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether (BRIJ®30)), poly (vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0336] Exemplary binding agents include, but are not limited to, starch (e.g. cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.

[0337] Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulation. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and / or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal.Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenyl ethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylatedhydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0338] In some embodiments, the pH of the pharmaceutical solutions are maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and / or sodium malate. In another embodiment, the exemplary buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not preferred due to complex formation and / or mRNA degradation.

[0339] Exemplary buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, etc., and / or combinations thereof.

[0340] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0341] Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, com, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils.Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0342] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0343] Exemplary additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents can be used.

[0344] In some embodiments of the present disclosure, the nucleic acid vaccine compositions described herein may comprise at least one nucleic acid vaccine that is formulated in a lipid nanoparticle (LNP) and at least one excipient. As non-limiting examples, the excipient may be a sugar such as sucrose. Formulations

[0345] In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid, or any combination thereof, wherein the neutral lipid, helper lipid, polymer-conjugated lipid are as defined herein. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid, at least one neutral lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid, at least one helper lipid, and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one neutral lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one helper lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one helper lipid and at least one polymer-conjugated lipid. In some embodiments, the LNP may be comprised of at least one ionizable lipid. In some embodiments, the LNP may be comprised of at least one neutral lipid. In some embodiments, a LNP may be comprised of a helper lipid. In some embodiments, the LNP may be comprised of a polymer-conjugated lipid. In some embodiments a lipid nanoparticle may be comprised of at least one ionizable lipid, at least one neutral lipid, at least one helper lipid, at least one polymer-conjugated lipid; and at least one hydrophobic component, wherein theionizable lipid, neutral lipid, helper lipid, polymer-conjugated lipid and hydrophobic component are as defined herein.

[0346] In some embodiments, the lipid nanoparticle can further comprise an adjuvant, a cell targeting component, a fat-soluble vitamin, an immunomodulating substance or a component that promotes absorption of drugs.

[0347] In some embodiments, the adjuvant with the lipid nanoparticle can be squalene, the cell targeting component can be cardiolipin, the fat-soluble vitamin can be vitamin A or E, the immunomodulating substance can be withaferin and the component that promote absorption of drugs can be caffeine.

[0348] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, can be from 0.1 to 100 mol%. In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), orthe lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), orthe lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), orthe compounds in Table 4 or Compound 109, in the lipid nanoparticle, is between 10%-95%, such as between about 10% to about 20%, between about 21% to about 30%, between about 31% to about 40%, between about 41% to about 50%, between about 51% to about 60%, between about 61%to about 70%, between about 71%to about 80%, between about 81% to about 90%, or between about 91% to about 95%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 40 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 20 to 60 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 50 to 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than about 60 mol% or about 85 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises an ionizable lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one ionizable lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0349] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), orthe lipid(s) having a structure of Formulas (II), (12), (13), (14), (15),(16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (He), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is from about 30% to 100%, from about 40% to 100%, from about 30% to about 90%, from about 30% to about 85%, from about 30% to about 80%, from about 30% to about 75%, from about 30% to about 70%, from about 30% to about 65%, or from about 30% to about 60%.

[0350] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 40% to about 90%, from about 40% to about 85%, from about 40% to about 80%, from about 40% to about 75%, from about 40% to about 70%, from about 40% to about 65%, or from about 40% to about 60%. In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (16a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is from about 40 to about 47 mol % of the ionizable lipid, preferably from about 40 to about 45 mol % of the ionizable lipid.

[0351] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18) or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 50% to about 90%, from about 50% to about 85%, from about 50% to about 80%, from about 50% to about 75%, from about 50% to about 70%, from about 50% to about 65%, or from about 50% to about 60%.

[0352] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 55% to about 90%, from about 55% to about 85%, from about 55% to about 80%, from about 55% to about 75%, from about 55% to about 70%, from about 55% to about 65%, or from about 55% to about 60%.

[0353] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a),(I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 60% to about 90%, from about 60% to about 85%, from about 60% to about 80%, from about 60% to about 75%, from about 60% to about 70%, or from about 60% to about 65%.

[0354] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 65% to about 90%, from about 65% to about 85%, from about 65% to about 80%, from about 65% to about 75%, or from about 65% to about 70%.

[0355] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 70% to about 90%, from about 70% to about 85%, from about 70% to about 80%, or from about 70% to about 75%.

[0356] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 75% to about 90%, from about 75% to about 85%, or from about 75% to about 80%.

[0357] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 80% to about 90%, or from about 80% to about 85%.

[0358] In some embodiments, the total mole percentage of the ionizable lipid, such as the lipid(s) having a structure of Formula (I), or the lipid(s) having a structure of Formulas (II), (12), (13), (14), (15), (16), (17) or (18), or the lipid(s) having a structure of Formulas (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a), or the compounds in Table 4 or Compound 109, in the lipid nanoparticle, is about 85% to about 90%.

[0359] In some embodiments, the ionizable lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0360] In some embodiments, transfer vehicle variability between lots can be less than 15%, less than 10% or less than 5%.

[0361] In some embodiments, when the lipid nanoparticle comprises at least one neutral lipid, the neutral lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 5 to 25 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of more than about 25 mol% or about 35 mol%. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one neutral lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiment, the lipid nanoparticle comprises at least one neutral lipid in an amount of at most bout 10 mol%.

[0362] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 18%, from about 5% to about 16%, from about 5% to about 14%, from about 5% to about 12%, from about 5% to about 10%, or from about 5% to about 8%.

[0363] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 18%, from about 7% to about 16%, from about 7% to about 14%, from about 7% to about 12%, from about 7% to about 10%, or from about 7% to about 8%.

[0364] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 18%, from about 9% to about 16%, from about 9% to about 14%, from about 9% to about 12%, or from about 9% to about 10%.

[0365] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 11% to about 20%, from about 11% to about 18%, from about 11% to about 16%, from about 11% to about 14%, or from about 11% to about 12%.

[0366] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 13% to about 20%, from about 13% to about 18%, from about 13% to about 16%, or from about 13% to about 14%.

[0367] In some embodiments, the total mole percentage of the neutral lipid in the lipid nanoparticle is about 15% to about 20%, or from about 17% to about 18%.

[0368] In some embodiments, the neutral lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0369] In some embodiments, when the lipid nanoparticle comprises at least one helper lipid, the helper lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of at most 50 mol%.In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 20 to 45 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 25 to 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than about 20 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than about 45 mol% or about 55 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one helper lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0370] In some embodiments, the helper lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0371] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 20% to about 50%, from about 20% to about 45%, from about 20% to about 40%, from about 20% to about 35%, from about 20% to about 30%, or from about 20% to about 25%.

[0372] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 25% to about 50%, from about 25% to about 45%, from about 25% to about 40%, from about 25% to about 35%, or from about 25% to about 30%.

[0373] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 30% to about 50%, from about 30% to about 45%, from about 30% to about 40%, or from about 30% to about 35%.

[0374] In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 35% to about 50%, from about 35% to about 45%, or from about 35% to about 40%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 40% to about 50%, or from about 40% to about 45%. In some embodiments, the total mole percentage of the helper lipid in the lipid nanoparticle is about 45% to about 50%.

[0375] In some embodiments, when the lipid nanoparticle comprises at least one polymer-conjugated lipid, the polymer-conjugated lipid can be present in the lipid nanoparticle in an amount of about 0.1 to 100 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 95 mol% or less. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 0.5 to 15 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of about 15 to 40 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than about 0.1 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of at most about 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of less than or equal to about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, and 5 mol%. In some embodiments, the lipid nanoparticle comprises at least one polymer-conjugated lipid in an amount of more than or equal to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mol%.

[0376] In some embodiments, the polymer-conjugated lipid mol % of the lipid nanoparticle can be ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the target mol %.

[0377] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.1% to about 10%, from about 0.1% to about 9%, from about 0.1% to about 8%, from about 0.1% to about 7%, from about 0.1% to about 6%, from about 0.1% to about 5%, or from about 0.1%to about 4%, from about 0.1%to about 3%, from about 0.1%to about 2%, or from about 0.1%to about 1%.

[0378] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 0.5% to about 10%, from about 0.5% to about 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, from about 0.5% to about 6%, from about 0.5% to about 5%, or from about 0.5% to about 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, or from about 0.5% to about 1%.

[0379] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 1% to about 10%, from about 1% to about 9%, from about 1% to about 8%, from about 1% to about 7%, from about 1% to about 6%, from about 1% to about 5%, from about 1% to about 4%, from about 1% to about 3%, or from about 1% to about 2%.

[0380] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 2% to about 10%, from about 2% to about 9%, from about 2% to about 8%, from about 2% to about 7%, from about 2% to about 6%, from about 2% to about 5%, from about 2% to about 4%, or from about 2% to about 3%.

[0381] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 3% to about 10%, from about 3% to about 9%, from about 3% to about 8%, from about 3% to about 7%, from about 3% to about 6%, from about 3% to about 5%, or from about 3% to about 4%.

[0382] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 4% to about 10%, from about 4% to about 9%, from about 4% to about 8%, from about 4% to about 7%, from about 4% to about 6%, or from about 4% to about 5%.

[0383] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 5% to about 10%, from about 5% to about 9%, from about 5% to about 8%, from about 5% to about 7%, or from about 5% to about 6%.

[0384] In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 6% to about 10%, from about 6% to about 9%, from about 6% to about 8%, or from about 6% to about 7%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 7% to about 10%, from about 7% to about 9%, or from about 7% to about 8%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the polymer-conjugated lipid in the lipid nanoparticle is about 8% to about 10%, or from about 8% to about 9%. In some embodiments, the total mole percentage of the PEG-lipid in the lipid nanoparticle is about 9% to about 10%.

[0385] In some embodiments, the lipid nanoparticle can comprise a hydrophobic component. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle at most about 20%, from about 1% to about 20%, from about 1% to about 15%, from about 1% to about 10%, or from about 1% to about 5%. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 3% to about 20%, from about 3% to about 15%, from about 3% to about 10%, or from about 3% to about 5%. In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is at most about 5 %.

[0386] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 5% to about 20%, from about 5% to about 15%, or from about 5% to about 10%.

[0387] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 7% to about 20%, from about 7% to about 15%, or from about 7% to about 10%.

[0388] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 9% to about 20%, from about 9% to about 15%, or from about 9% to about 10%.

[0389] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 11% to about 20%, or from about 11% to about 15%.

[0390] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about 13% to about 20%, or from about 13% to about 15%.

[0391] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 15% to about 20%.

[0392] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 17% to about 20%.

[0393] In some embodiments, the total mole percentage of the hydrophobic component in the lipid nanoparticle is about or from about 19% to about 20%.

[0394] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0395] The at least one ionizable lipid is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie). (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof.

[0396] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0397] In some embodiments, the lipid nanoparticle is comprised of about 35-45 mol% of at least one ionizable lipid, about 25-35 mol% of at least one neutral lipid (e.g., a phospholipid), about 20-30 mol% of at least one helper lipid (e.g., cholesterol), and about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0398] In some embodiments, the lipid nanoparticle is comprised of about 45-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0399] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid, about 5-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 35-45 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-3 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0400] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 15-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.01-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0401] In some embodiments, the lipid nanoparticle is comprised of about 10-75 mol% of at least one ionizable lipid, about 0.5-50 mol% of at least one neutral lipid (e.g., a phospholipid), about 5-60 mol% of at least one helper lipid (e.g., cholesterol), and about 0.1-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0402] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0403] In some embodiments, the lipid nanoparticle is comprised of about 50-85 mol% of at least one ionizable lipid, about 3-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-40 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-2 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0404] In some embodiments, the lipid nanoparticle is comprised of about 25-75 mol% of at least one ionizable lipid, about 0.1-15 mol% of at least one neutral lipid (e.g., a phospholipid), about 5-50 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-20 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0405] In some embodiments, the lipid nanoparticle is comprised of about 50-65 mol% of at least one ionizable lipid, about 5-10 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-35 mol% of at least one helper lipid (e.g., cholesterol), and about 5-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0406] In some embodiments, the lipid nanoparticle is comprised of about 20-60 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 25-55 mol% of at least one helper lipid (e.g., cholesterol), and about 0.5-15 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid).

[0407] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0408] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0409] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid(e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0410] In some embodiments, the lipid nanoparticle is comprised of about 30-60 mol% of at least one ionizable lipid, about 0-30 mol% of at least one neutral lipid (e.g., a phospholipid), about 18.5-48.5 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0411] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-20 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0412] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-15 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0413] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 0-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0414] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid, about 5-25 mol% of at least one neutral lipid (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0415] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) andabout 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0416] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, -carotene, and / or a-tocopherol.

[0417] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol.

[0418] In some embodiments, the lipid nanoparticle is comprised of about 35-55 mol% of at least one ionizable lipid selected from Table 4 or Compound 109 herein, about 5-25 mol% of at least one neutral (e.g., a phospholipid), about 30-45 mol% of at least one helper lipid (e.g., cholesterol), about 0-10 mol% of at least one polymer-conjugated lipid (e.g., a PEG-lipid) and about 4-10 mol% of at least one hydrophobic component (e.g., squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol).

[0419] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 4 or Compound 109 herein, about 5-10 mol% of at least one phospholipid or phospholipid derivative described herein, about 30-45 mol% of at least one cholesterol or cholesterol derivative described herein, about 1-4 mol% of at least one PEG-lipid described herein and about 0-5 mol% of squalene, cardiolipin, withaferin A, vitamin A, retinol, P-carotene and / or a-tocopherol.

[0420] In some embodiments, the lipid nanoparticle is comprised of about 40-60 mol% of at least one ionizable lipid selected from Table 4 or Compound 109 herein, about 5-10 mol% of DSPC, about 30-45 mol% of cholesterol, about 1-4 mol% of PEG-DMG and about 0-5 mol% of squalene, cardiolipin, vitamin A, retinol, P-carotene, withaferin A and / or a-tocopherol.

[0421] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 100 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b) (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as definedherein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from 0 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0422] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 99 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from 0 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0423] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0424] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from 0 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0425] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from 0 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0426] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 1 to about 10 mol % of the neutral lipid; from about 1 to about 50 mol % of the helper lipid; from about 1 to about 5 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0427] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 50 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from about 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0428] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of the neutral lipid; from about 30 to about 45 mol % of the helper lipid; from about 1 to about 4 mol % of the polymer-conjugated lipid; and from 0.1 to about 5 mol % of the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0429] In some embodiments, the lipid nanoparticle can comprise from about 40 to about 60 mol % of the ionizable lipid which is at least one compound having a structure of Formula (I), (II), (12), (13), (14), (15), (16), (17), (18), (Ila), (lib), (lie), (I2a), (I2b), (I3a), (I4a), (I5a), (I6a), (I7a) or (I8a) as defined herein, or at least one compound in Table 4 or Compound 109 herein, or a pharmaceutically acceptable salt thereof; from about 5 to about 10 mol % of a phospholipid as the neutral lipid; from about 30 to about 50 mol % of a sterol as the helper lipid; from about 1 to about 4 mol % of a PEG-lipid as the polymer-conjugated lipid; and from 0.1 to about 5 mol % of squalene, cardiolipin, a-tocopherol, withaferin A, vitamin A, or a combination thereof as the hydrophobic component; wherein the mol % are based on the total lipids present in the nanoparticle.

[0430] In some embodiments, the lipid nanoparticle can comprise an ionizable lipid (Compound 201, 44% mol), a neutral lipid (DSPC, 10% mol), cholesterol (39% mol), a polymer-conjugated lipid (PEG2k-DMG, 2% mol) and a hydrophobic component (Squalene, 5% mol). The nucleic acid vaccine may be formulated in said lipid nanoparticle, and the formulation may also comprise a cryoprotectant (e.g., sucrose), a buffer (e.g., tris (tromethamine)) and / or a solvent or vehicle (e.g., water). To achieve the preferred dose nucleic acid vaccine, the nucleic acid vaccine may be diluted in an isotonic sodium chloride solution (e.g., 09.%NaCl, saline).Encapsulation

[0431] In some embodiments, the lipid nanoparticle may fully or partially encapsulate a nucleic acid vaccine, e.g., a mRNA vaccine. In some embodiments, essentially 0% of the nucleic acid vaccine present in the final formulation is exposed to the environment outside of the lipid nanoparticle (i.e., the nucleic acid vaccine is fully encapsulated. In some embodiments, the nucleic acid vaccine is associated with the lipid nanoparticle but is at least partially exposed to the environment outside of the lipid nanoparticle. In some embodiments, the lipid nanoparticle may be characterized by the % of the nucleic acid vaccine not exposed to the environment outside of the lipid nanoparticle, e.g., the encapsulation efficiency. For the sake of clarity, an encapsulation efficiency of about 100% refers to a lipid nanoparticle formulation where essentially all the nucleic acid vaccine is fully encapsulated by the lipid nanoparticle, while an encapsulation rate of about 0% refers to a lipid nanoparticle where essential none of the nucleic acid vaccine is encapsulated in the lipid nanoparticle, such as with a lipid nanoparticle where the nucleic acid vaccine is bound to the external surface of the lipid nanoparticle. On some embodiments, a lipid nanoparticle may have an encapsulation efficiency of less than about 100%, less than about 95%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15% less than about 10%, or less than 5%. In some embodiments, an lipid nanoparticle may have an encapsulation efficiency of between about 90 to 100%, 80 to 100%, 70 to 100%, 60 to 100%, 50 to 100%, 40 to 100%, 30 to 100%, 20 to 100%, 10 to 100%, 80 to 90%, 70 to 90%, 60 to 90%, 50 to 90%, 40 to 90%, 30 to 90%, 20 to 90%, 10 to 90%, 70 to 80%, 60 to 80%, 50 to 80%, 40 to 80%, 30 to 80%, 20 to 80%, 10 to 80%, 60 to 70%, 50 to 70%, 40 to 70%, 30 to 70%, 20 to 70%, 10 to 70%, 40 to 50%, 30 to 50%, 20 to 50%, 10 to 50%, 30 to 40%, 20 to 40%, 10 to 40%, 20 to 30%, 10 to 30%, and 10 to 20%.

[0432] In some embodiments, the weight ratio of the lipid nanoparticle (including all the lipids) and the nucleic acid vaccine is between about 100: 1 to about 1:1, such as between about 100: 1 to about 90: 1, between about 89: 1 to about 80: 1, between about 79: 1 to about 70: 1, between about 69: 1 to about 60: 1,between about 59: 1 to about 50:1, between about 49: 1 to about 40: 1, between about 39: 1 to about 30:1, between about 29: 1 to about 20: 1, between about 19: 1 to about 10:1, and between about 9: 1 to about 1:1.

[0433] In some embodiments, the at least one nucleic acid vaccine is comprised of a functional RNA where the result is at least one change in a cell, tissue, organ and / or organism. Said changes in state may include, but are not limited to, altering the expression level of a polypeptide, altering the translation level of a nucleic acid, altering the expression level of a nucleic acid, altering the amount of a polypeptide present in a cell, tissue, organ and / or organism, changing a genetic sequence of a cell, tissue, organ and / or organism, adding nucleic acids to a target genome, subtracting nucleic acids from a target genome, altering physiological activity in a cell, tissue, organ and / or organism or any combination thereof.Methods of Preparation of Lipid Nanoparticles

[0434] The lipid nanoparticles described herein may be formed using techniques known in the art. As a non-limiting example, an organic solution containing the lipids is mixed together with an acidic aqueous solution containing the originator construct or benchmark construct in a microfluidic channel resulting in the formation of targeting system (lipid nanoparticle and the benchmark construct).

[0435] In some embodiments, the lipid nanoparticle formulations disclosed herein may be prepared by the methods described in International Publication No W02008103276, the content of which is herein incorporated by reference in its entirety. In some embodiments, lipid nanoparticle formulations may be as described in International Publication No. W02019131770, the content of which is herein incorporated by reference in its entirety.

[0436] In some embodiments, a lipid nanoparticle formulation may be prepared by the methods described in International Publication No. WO2020237227, the content of which is herein incorporated by reference in its entirety.

[0437] In some embodiments, a lipid nanoparticle formulations disclosed herein may be prepared using the methodologies and devices described in US20240181406A1, the content of which is herein incorporated by reference in its entirety.

[0438] In further embodiments, a lipid nanoparticle formulations disclosed herein may be prepared using the FDmiX mixing systems developed by FDX Fluid Dynamix GmbH (See https: / / www.fdx.de / en / fdmix / , the content of which is herein incorporated by reference in its entirety).

[0439] In some embodiments, the lipid nanoparticle formulations disclosed herein may be prepared by a so-called “Point-of-Care” mixing method, which consists in preparing separately a solution of a cargo, such as mRNA for instance, that can be frozen or lyophilized for storage, and a solution of lipid. Mixing the lipid solution and the mRNA solution via transferring the mRNA to a sealed vial containing the lipid followed by vigorous shaking of the vial provides mRNA encapsulated in LNPs ready for using / dosing. Point-of-Care mixing can produce mRNA-LNPs with comparable quality and efficacy tomRNA-LNPs obtained with conventional methods (conventional mRNA-LNPs). Manufacturing of conventional mRNA-LNPs is a sensitive process and usually involves specialized equipment and analytical techniques. The mRNA-LNPs are sensitive to minor changes in the manufacturing process. Long-term storage of mRNA-LNPs requires cryo-storage facilities. Still long-term stability of mRNA-LNPs may not be ensured. On the other hand, in Point-of-Care mixing, the LNPs are not manufactured ahead of time of administration. The mRNA and lipid solution can be manufactured, fdled and released using conventional small-molecule pharmaceutical manufacturing units. Both the mRNA and lipid solution are stable under frozen conditions.

[0440] In some embodiment, the lipid nanoparticle formulations disclosed herein may be prepared from a nucleic acid vaccine that has been stored frozen (-80 + / - 10 °C) and then thawed and diluted with an isotonic sodium chloride solution (0.9% NaCl, saline) to achieve the targeted dose of the nucleic acid vaccine.

[0441] In some embodiments, administered to a subject occurs within 4 hours of preparing the formulation nucleic acid vaccine. Not to be bound by theory, it is predicted that the short lead time is in order to minimize the risk of microbial contamination. The conditions during this preparation, handling and transfer may be room temperature (about 20-23 + / - 2°C) and then refrigeration (about 2-8°C) for short-term storage before administration to a subject.Adjuvants

[0442] Adjuvants may also be administered with or in combination with one or more of the nucleic acid vaccines described herein, e.g., the mRNA vaccine. Adjuvants may be used to enhance the immunogenicity of the nucleic acid vaccine, modify the immune response, reduce the amount of nucleic acid vaccine needed for immunization, reduced the frequency of additional or “booster” immunizations needed or to create an improved immune response in those with weakened or immunocompromised immune systems or the elderly. The adjuvants may be a component of the formulation containing the nucleic acid vaccine or they may be co-administered with the nucleic acid vaccines compositions. Coadministration of the adjuvant may be any method known in the art or described herein such as, but not limited to, intravenous (IV), intramuscular (IM), subcutaneous (SC) or intradermal (ID).

[0443] In some embodiments, the adjuvant is natural or synthetic. The adjuvants may also be organic or inorganic.

[0444] In some embodiments, the adjuvant used with the nucleic acid vaccine is from a class of adjuvants such as, but not limited to carbohydrates, microorganisms, mineral salts (e.g., aluminum hydroxide, aluminum phosphate gel, or calcium phosphate gel), emulsions (e.g., oil emulsion, surfactant based emulsion, purified saponin, and oil-in water emulsion), inert vehicles, particulate adjuvants (e.g.,unilamellar liposomal vehicles such as virosomes or a structured complex of saponions and lipids such as polylactide co-glycolide (PLG)), microbial derivatives, endogenous human immunomodulators, and tensoactive compounds. Listings of adjuvants which may be used with the nucleic acid vaccines described herein may be found on the web-based vaccine adjuvant database Vaxjo (see e.g., violinet.org / vaxjo or Sayers et al., Journal of Biomedicine and Biotechnology 2012; 2012:831486.. PMID: 22505817; the contents of which are herein incorporated by reference in their entirety).

[0445] Adjuvants may be selected for use with the nucleic acid vaccines by one of ordinary skill in the art. Adjuvants may be interferons, TNF-alpha, TNF-beta, chemokines (e.g., CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD40L, CD28 agonists, PD1, soluble PD1, PDL1, PDL2) or interleukins (e g., IL1, IL2, IL4, IL6, IL7, IL10, IL12, IL13, IL15, IL 17, IL 18, IL21, and IL23). Non-limiting examples of adjuvants include Abisco-100 vaccine adjuvant, Adamantyl amide Dipeptide Vaccine Adjuvant, Adjumer™, AF03, Albumin-heparin microparticles vaccine adjuvant, Algal Glucan, Algammulin, alhydrogel, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, Aluminum vaccine adjuvant, amorphous aluminum hydroxyphosphate sulfate adjuvant, Arlacel A, ASO, AS04, AS03, AS-2 vaccine adjuvant, Avridine®, B7-2 vaccine adjuvant, Bay R1005, Bordetella pertussis component Vaccine Adjuvant, Bupivacaine vaccine adjuvant, Calcium Phosphate Gel, Calcium phosphate vaccine adjuvant, Cationic Liposomal Vaccine Adjuvant, cationic liposome-DNA complex JVRS- 100, Cholera toxin, Cholera toxin B subunit, Corynebacterium-derived P40 Vaccine Adjuvant, CpG DNA Vaccine Adjuvant, CRL1OO5, CTA1-DD gene fusion protein, DDA Adjuvant, DHEA vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, DOC / Alum Complex, E. coli heat-labile toxin, Etx B subunit Adjuvant, Flagellin, Freund’s Complete Adjuvant, Freund’s Incomplete Adjuvant, Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Imiquimod, Immunoliposomes Containing Antibodies to Costimulatory Molecules, ISCOM(s)™, ISCOMA-TRIX®, Killed Corynebacterium parvum Vaccine Adjuvant, Lipopolysaccharide, Liposomes, Loxoribine, LTK63 Vaccine Mutant Adjuvant, LTK72 vaccine adjuvant, LTR192G Vaccine Adjuvant, Matrix-S, MF59, Montanide Incomplete Seppic Adjuvant, Montanide ISA 51, Montanide ISA 720 Adjuvant, MPL-SE vaccine adjuvant, MPL™ Adjuvant, MTP-PE Liposomes, Murametide, Muramyl Dipeptide Adjuvant, Murapalmitine, D-Murapalmitine, NAGO, nanoemulsion vaccine adjuvant, Non-Ionic Surfactant Vesicles, non-toxic mutant El 12K of Cholera Toxin mCT-El 12K, PMMA, Poly(LC), Polygen Vaccine Adjuvant, Protein Cochleates, QS-21, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, Recombinant hlFN-gamma / Interferon-g, Rehydragel EV, Rehydragel HPA, Resiquimod, Ribi Vaccine Adjuvant, SAF-I, Saponin Vaccine Adjuvant, Sclavo peptide, Sendai Proteoliposomes, Sendai -containing Lipid Matrices, Specol, SPT (Antigen Formulation), Squalene-based Adjuvants, Stearyl Tyrosine, Theramide®, Threonylmuramyl dipeptide (TMDP), Titer-Max Gold Adjuvant, Ty Particles vaccine adjuvant, and VSA-3 Adjuvant.

[0446] In some embodiments, the nucleic acid vaccines described herein may be used as a vaccine and may further comprise an adjuvant which may enable the vaccine to elicit a higher immune response. As a non-limiting example, the adjuvant could be a sub-micron oil-in-water emulsion which can elicit a higher immune response in human pediatric populations (see e.g., the adjuvanted vaccines described in US Patent Publication No. US20120027813 and U.S. Pat. No. 8,506,966, the contents of each of which are herein incorporated by reference in their entirety).Administration Routes

[0447] The lipid nanoparticles comprising payloads and / or pharmaceutical compositions described herein may be administered by any delivery route which results in a therapeutically effective outcome. In some embodiments, lipid nanoparticles comprising payloads and / or pharmaceutical compositions described herein may be administered parenterally. Uiquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and / or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof. In other embodiments, surfactants are included such as hydroxypropylcellulose.

[0448] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed includingsynthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0449] Injectable formulations may be sterilized, for example, by fdtration through a bacterial-retaining fdter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0450] In order to prolong the effect of active ingredients, it is often desirable to slow the absorption of active ingredients from subcutaneous or intramuscular injections. This may be accomplished by the use of liquid suspensions of crystalline or amorphous material with poor water solubility. The rate of absorption of active ingredients depends upon the rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactidepolyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.

[0451] Formulations may also be delivered to a patient via intranasal administration.Dosins and Administration

[0452] The present disclosure encompasses the delivery of nucleic acid vaccine compositions including, for example, nucleic acid vaccine for any therapeutic, prophylactic (including post-exposure and pre-exposure), pharmaceutical, diagnostic or imaging use by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.

[0453] The nucleic acid vaccine compositions of the present disclosure may be delivered to a cell naked. As used herein in, “naked” refers to delivering nucleic acid vaccine compositions free from agents which promote transfection. For example, the nucleic acid vaccine compositions delivered to the cell may contain no modifications. The naked nucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.

[0454] The nucleic acid vaccine compositions of the present disclosure may be formulated, using the formulation components and methods described herein. The formulations may contain nucleic acid vaccine compositions which may be modified and / or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulatednucleic acid vaccine compositions may be delivered to the cell using routes of administration known in the art and described herein.

[0455] The nucleic acid vaccine compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and / or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like. The nucleic acid vaccine compositions of the present disclosure may also be cloned into a retroviral replicating vector (RRV) and transduced to cells.Dosins

[0456] Provided herein also include methods comprising administering the nucleic acid vaccines described herein to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, health, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0457] The present disclosure contemplates dosage levels of between about 0.001 and about 500 mg nucleic acid vaccine / kg body weight, about 0.001 and about 200 mg / kg, about 0.001 and about 100 mg / kg, 0.01 and about 100 mg / kg, preferably between about 0.005 and about 50 mg / kg, 0.01 and about 50 mg / kg, 0.01 and about 40 mg / kg, 0.01 and about 30 mg / kg, 0.01 and about 10 mg / kg, 0.05 and about 50 mg / kg, 0.05 and about 30 mg / kg, 0.05 and about 10 mg / kg, 0.05 and about 5 mg / kg, 0.1 and about 50 mg / kg, 0.1 and about 30 mg / kg, 0.1 and about 10 mg / kg, 0.1 and about 1 mg / kg, 1.0 and about 50 mg / kg, 1.0 and about 40 mg / kg, 1.0 to about 30 mg / kg, 10 to about 50mg / kg body weight. Other embodiments contemplate a dosage of between about 0.001-0.010, 0.010-0.050, 0.050-0.100, 0.1-0.5, 0.5-1.0, 1.0-5.0, 5.0-10, 10-50 mg / kg, 10-100mg / kg body weight. The dosages may be administered about hourly, multiple times per day, daily, every other day, weekly, every other week, monthly, every other month, or on an as-needed basis.

[0458] In some embodiments, compositions of the nucleic acid vaccines may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, from about 1 mg / kg to about 25 mg / kg, from about 1 mg / kg to about 50 mg / kg, from about 10 mg / kg to about 100 mg / kg, from about 10 mg / kg to about 50 mg / kg, of subject body weight, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

[0459] In some embodiments, compositions of the nucleic acid vaccines described herein may be administered at dosage levels sufficient to deliver to a subject, about 1 pg, lOpg, 15pg, 20pg, 25pg, 30pg, 35pg, 40pg, 50pg, 60pg, 70pg, 80pg, 90pg, or lOOpg of the nucleic acid composition.

[0460] In some embodiments, compositions of the nucleic acid vaccines may be administered at dosage levels sufficient to deliver from about 1-5 pg, 1-10 pg, 1-15 pg, 1-20 pg, 1-25 pg, 1-50 pg, 5-10 pg, 5-15 pg, 5-20 pg, 5-25 pg, 5-50 pg, 10-15 pg, 10-20 pg, 10-25 pg, 10-50 pg, 15-20 pg, 20-25 pg, 20-50 pg, 20-60 pg, 20-70 pg, 20-80 pg, 20-90 pg, 20-100 pg, 20-200 pg, 20-300 pg, 20-400 pg, 20-500 pg, 20-600 pg, 20-700 pg, 20-800 pg, 20-900 pg, 20-1000 pg, 25-50 pg, 25-60 pg, 25-70 pg, 25-80 pg, 25-90 pg, 25-100 pg, 25-200 pg, 25-300 pg, 25-400 pg, 25-500 pg, 25-600 pg, 25-700 pg, 25-800 pg, 25-900 pg, 25-1000 pg, 30-50 pg, 30-60 pg, 30-70 pg, 30-80 pg, 30-90 pg, 30-100 pg, 30-200 pg, 30-300 pg, 30-400 pg, 30-500 pg, 30-600 pg, 30-700 pg, 30-800 pg, 30-900 pg, 30-1000 pg, 40-50 pg, 40-60 pg, 40-70 pg, 40-80 pg, 40-90 pg, 40-100 pg, 40-200 pg, 40-300 pg, 40-400 pg, 40-500 pg, 40-600 pg, 40-700 pg, 40-800 pg, 40-900 pg, 40-1000 pg, 50-60 pg, 50-70 pg, 50-80 pg, 50-90 pg, 50-100 pg, 50-200 pg, SO-SOO pg, 50-400 pg, 50-500 pg, 50-600 pg, 50-700 pg, 50-800 pg, 50-900 pg, 50-1000 pg, 60-70 pg, 60-80 pg, 60-90 pg, 60-100 pg, 60-200 pg, 60-300 pg, 60-400 pg, 60-500 pg, 60-600 pg, 60-700 pg, 60-800 pg, 60-900 pg, 60-1000 pg, 70-80 pg, 70-90 pg, 70-100 pg, 70-200 pg, 70-300 pg, 70-400 pg, 70-500 pg, 70-600 pg, 70-700 pg, 70-800 pg, 70-900 pg, 70-1000 pg, 80-90 pg, 80-100 pg, 80-200 pg, 80-300 pg, 80-400 pg, 80-500 pg, 80-600 pg, 80-700 pg, 80-800 pg, 80-900 pg, 80-1000 pg, 90-100 pg, 90-200 pg, 90-300 pg, 90-400 pg, 90-500 pg, 90-600 pg, 90-700 pg, 90-800 pg, 90-900 pg, 90-1000 pg, 100-200 pg, 100-300 pg, 100-400 pg, 100-500 pg, 100-600 pg, 100-700 pg, 100-800 pg, 100-900 pg, 100-1000 pg,150-200 pg, 150-300 pg, 150-400 pg, 150-500 pg, 150-600 pg, 150-700 pg, 150-800 pg, 150-900 pg, 150-1000 pg, 200-300 pg, 200-400 pg, 200-500 pg, 200-600 pg, 200-700 pg, 200-800 pg, 200-900 pg, 200-1000 pg, 250-300 pg, 250-400 pg, 250-500 pg, 250-600 pg, 250-700 pg, 250-800 pg, 250-900 pg, 250-1000 pg, 300-400 pg, 300-500 pg, 300-600 pg, 300-700 pg, 300-800 pg, 300-900 pg, 300-1000 pg, 350-400 pg, 350-500 pg, 350-600 pg, 350-700 pg, 350-800 pg, 350-900 pg, 350-1000 pg, 400-500 pg, 400-600 pg, 400-700 pg, 400-800 pg, 400-900 pg, 400-1000 pg, 450-500 pg, 450-600 pg, 450-700 pg, 450-800 pg, 450-900 pg, 450-1000 pg, 500-600 pg, 500-700 pg, 500-800 pg, 500-900 pg, 500-1000 pg, 550-600 pg, 550-700 pg, 550-800 pg, 550-900 pg, 550-1000 pg, 600-700 pg, 600-800 pg, 600-900 pg, 600-1000 pg, 650-800 pg, 650-900 pg, 650-1000 pg, 700-800 pg, 700-900 pg, 700-1000 pg, 750-800 pg, 750-900 pg, 750-1000 pg, 800-900 pg, 800-1000 pg, 850-900 pg, 850-1000 pg, 900-1000 pg, or 950-1000 pg.

[0461] In some embodiments, the nucleic acid vaccines may be administered in split-dose regimens. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g., two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24-hour period. It may be administered as a single unit dose. In some embodiments, the nucleic acid vaccines described herein are administered to a subject in split doses. The nucleic acid vaccines may be formulated in buffer only or in a formulation described herein.

[0462] In some embodiments, the nucleic acid vaccine compositions described herein may be administered to a subject in two separate phases, a loading dosing phase and a maintenance dosing phase. The dosing regimen may comprise an initial higher loading dose of the nucleic acid vaccine that is given to the subject first time at the beginning of a course of prevention, alleviation and / or treatment, e.g., first dose for preventing, and a lower maintenance dose following the first loading dose. In some embodiments, the loading dose and the maintenance dose have the same amount of the nucleic acid vaccines of the present disclosure. In some embodiments, more than one maintenance doses are administered to the subject. The multiple maintenance doses may be administered biweekly, every three weeks, every four weeks, monthly, bimonthly, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every 11 months or every 12 months. In the context of vaccination for prevention of a disorder, the maintenance doses of the nucleic acid vaccines may also be referred to as booster doses. As used herein, a “booster dose” (or “booster shot”) is an extra or supplemental dose of a vaccine after an initial primer dose. The booster dose may have the same amount of the nucleic acid vaccine as the initial loading dose.Alternatively, the booster dose has an amount of the nucleic acid vaccine that is smaller than the originalamount of the nucleic acid vaccine in the initial dose. Alternatively, the booster dose has an amount of the nucleic acid vaccine that is larger than the original amount of the nucleic acid vaccine in the initial dose. In some embodiments, the subject may receive one, two, three, four or more booster doses. In some embodiments, the booster dose may be administered at least 1 month after the initial primer dose. In some embodiments, the booster dose may be administered at least 2 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 3 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 4 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 5 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 6 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 7 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 8 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 9 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 10 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 11 months after the initial primer dose. In some embodiments, the booster dose may be administered at least 12 months after the initial primer dose. In some embodiments, the booster dose may be administered yearly after the initial primer dose. In some embodiments, the booster dose may be administered every 2 years after the initial primer dose. In some embodiments, the booster dose may be administered every 3 years after the initial primer dose. In some embodiments, the booster dose may be administered every 4 years after the initial primer dose. In some embodiments, the booster dose may be administered every 5 years after the initial primer dose. In some embodiments, the booster dose may be administered every 6 years after the initial primer dose. In some embodiments, the booster dose may be administered every 7 years after the initial primer dose. In some embodiments, the booster dose may be administered every 8 years after the initial primer dose. In some embodiments, the booster dose may be administered every 9 years after the initial primer dose. In some embodiments, the booster dose may be administered every 10 years after the initial primer dose.

[0463] Such administration can be used as a chronic or acute treatment or prevention of a clinicconcerning condition. The amount of drug that may be combined with the carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. A typical preparation will contain from about 5% to about 95% active compound (w / w). Preferably, such preparations contain from about 20% to about 80%, 30% to about 70%, 40% to about 60%, or about 50% active compound. In other embodiments, the preparations used in the present disclosure will include 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-99%, or greater than 99% of the active ingredient.

[0464] Upon improvement of a patient's condition, a maintenance dose of a compound, composition or combination of the present disclosure may be administered, if necessary. Subsequently, the dosage or frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained when the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment on a long-term basis upon any recurrence of disease symptoms.

[0465] As the skilled artisan will appreciate, lower or higher doses than those recited above may be required. Specific dosage and treatment regimens for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health status, gender, diet, time of administration, rate of excretion, drug combination, the severity and course of an infection, the patient's disposition to the infection and the judgment of the treating physician.Delivery

[0466] In some embodiments, the delivery of the nucleic acid vaccines may be naked or formulated.

[0467] In some embodiments, the nucleic acid vaccines described herein may be delivered to a cell naked. As used herein in, “naked” refers to delivering nucleic acid vaccines free from agents which promote transfection. For example, the nucleic acid vaccines delivered to the cell may contain no modifications. The naked nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.

[0468] In some embodiments, the nucleic acid vaccines described herein may be formulated, using the methods described herein. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated nucleic acid vaccines may be delivered to the cell using routes of administration known in the art and described herein.

[0469] The compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and / or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.Ill, METHODS OF USE

[0470] One aspect of the present disclosure provides methods of using nucleic acid vaccines of the present disclosure and pharmaceutical compositions and formulations comprising the nucleic acid vaccines and at least one pharmaceutically acceptable carrier. Provided herein are compositions, methods, kits, and reagents for diagnosis, treatment, alleviation or prevention of a disease or condition in humans orother mammals where the active agent is the nucleic acid vaccine, cells containing the nucleic acid vaccine or polypeptides translated from nucleic acid vaccine polynucleotides.

[0471] In some embodiments, the methods of use can be assessed using any endpoint indicating a benefit to the subject, including, without limitation, (1) inhibition, to some extent, of disease progression, including stabilization, slowing down and complete arrest; (2) reduction in the number of disease episodes and / or symptoms; (3) inhibition ( .e., reduction, slowing down or complete stopping) of a disease cell infiltration into adjacent peripheral organs and / or tissues; (4) inhibition ( .e. reduction, slowing down or complete stopping) of disease spread; (5) decrease of an autoimmune condition; (6) favorable change in the expression of a biomarker associated with the disorder; (7) relief, to some extent, of one or more symptoms associated with a disorder; (8) increase in the length of disease-free presentation following treatment; or (9) decreased mortality at a given point of time following treatment. Cancers

[0472] In some embodiments, the lipid nanoparticles comprising payloads cargos / payloads described herein may be used for treating cancer. Hence, in some embodiments, the lipid nanoparticles can comprise an anti-cancer cargo or a cargo triggering an immune response against cancer cells. According to the present disclosure, cancer embraces any disease or malady characterized by uncontrolled cell proliferation, e.g., hyperproliferation. Cancers may be characterized by tumors, e.g., solid tumors or any neoplasm.

[0473] In some embodiments, the lipid nanoparticles comprising cargos / payloads of the present disclosure have been found to inhibit cancer and / or tumor growth. They may also reduce, including cell proliferation, invasiveness, and / or metastasis, thereby rendering them useful for the treatment of a cancer.

[0474] In some embodiments, the lipid nanoparticles comprising cargos / payloads of the present disclosure may be used to prevent the growth of a tumor or cancer, and / or to prevent the metastasis of a tumor or cancer. In some embodiments, compositions of the present teachings may be used to shrink or destroy a cancer.

[0475] In some embodiments, the lipid nanoparticles comprising cargos / payloads provided herein are useful for inhibiting proliferation of a cancer cell. In some embodiments, the lipid nanoparticles comprising payloads provided herein are useful for inhibiting cellular proliferation, e.g., inhibiting the rate of cellular proliferation, preventing cellular proliferation, and / or inducing cell death. In general, the lipid nanoparticles comprising cargos / payloads as described herein can inhibit cellular proliferation of a cancer cell or both inhibiting proliferation and / or inducing cell death of a cancer cell.

[0476] The cancers treatable by methods of the present teachings generally occur in mammals.Mammals include, for example, humans, non-human primates, dogs, cats, rats, mice, rabbits, ferrets, guinea pigs, horses, pigs, sheep, goats, and cattle. In various embodiments, the cancer is lung cancer,breast cancer, e.g., mutant BRCA1 and / or mutant BRCA2 breast cancer, non-BRCA-associated breast cancer, colorectal cancer, ovarian cancer, pancreatic cancer, colorectal cancer, bladder cancer, prostate cancer, cervical cancer, renal cancer, leukemia, central nervous system cancers, myeloma, and melanoma.

[0477] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of basal cell carcinomas.

[0478] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of bladder cancer, also known as urothelial cancer.

[0479] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of bile duct cancer, also known as cholangiocarcinoma.

[0480] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of breast cancer.

[0481] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of cervical cancer. As a non-limiting example, the cervical cancer may be HPV negative.

[0482] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of colorectal cancer, which is also known as colon cancer.

[0483] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of endometrial cancer.

[0484] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of esophageal cancer.

[0485] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of female reproductive system-associated carcinomas (e.g., endometrial and uterine serous carcinomas).

[0486] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of gastric cancer.

[0487] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of glioblastoma.

[0488] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of glioma.

[0489] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of head and neck cancer. As a non-limiting example, the head and neck cancer is head and neck squamous cell carcinoma (HNSCC).

[0490] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of hepatocellular cancer (HCC) which is also known as liver cancer.

[0491] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of kidney cancer.

[0492] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of leukemia. As a non-limiting example, the leukemia is acute myeloid leukemia (AML).

[0493] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of lymphoma. As a non-limiting example, the lymphoma is diffuse large B cell lymphoma (DLBCL).

[0494] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of lung cancer. As a non-limiting example, the lung cancer is non-small cell lung cancer (NSCLC).

[0495] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of melanoma. As a non-limiting example, the melanoma is an advanced melanoma. As a non-limiting example, the melanoma is an uveal melanoma. As a non-limiting example, the melanoma is metastatic melanoma. As a non-limiting example, the melanoma is cutaneous melanoma.

[0496] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of multiple myeloma.

[0497] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of mesothelioma.

[0498] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of nervous system -associated tumors.

[0499] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of neuroblastoma.

[0500] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of ovarian cancer. As a non-limiting example, the ovarian cancer is epithelial ovarian cancer.

[0501] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of ovarian clear cell carcinomas.

[0502] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of pancreatic cancer. As a non-limiting example, the pancreatic cancer is pancreatic adenocarcinoma (also referred to as ductal carcinoma).

[0503] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of prostate cancer.

[0504] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of renal cell carcinoma.

[0505] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of sarcomas. As a non-limiting example, the sarcoma is synovial sarcoma. As a non-limiting example, the sarcoma is soft tissue sarcoma. As a nonlimiting example, the sarcoma is osteosarcoma. As a non-limiting example, the sarcoma is dedifferentiated liposarcoma.

[0506] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of thymic carcinomas.

[0507] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of ependymoma.

[0508] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of diffuse midline glioma (DMG).

[0509] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of medulloblastoma.

[0510] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, are used for the prevention, alleviation and / or treatment of medullary thyroid cancer (MTC).Glioblastoma (GBM)

[0511] Glioblastoma, also known as glioblastoma multiforme, (GBM) is the most common type of malignant brain tumor or cancerthat originates in the brain and / or spinal cord. Rarely, the cancer spreads outside the brain to other parts of the body. It is an aggressive type of cancer that is often characterized by its rapid growth due to glioblastoma cancer cells growing and multiplying quickly. There is currently no cure for the condition and the prognosis for patients is not good and often less than six months as GBM is usually resistant to the current treatments and it has a high likelihood of recurrence. Current treatments focus on removing or shrinking the tumor to reduce the symptoms, to extend the life or improve the quality of life. While glioblastoma most often affects people aged 45 to 70 years old, the averagediagnosed age is closer to 64 years old but the disease affects all ages including children. Men and people assigned male at birth have a slightly higher risk, but the disease affects is not limited to them.

[0512] Healthcare providers use a grading system from I (1) to IV (4) to indicate brain tumor behavior where grade 1 (1) tumors grows slowly and are the least aggressive and grade IV (4) tumors grow rapidly and are more aggressive. GBM tumors are grade IV (4) by definition.

[0513] Glioblastoma can also be classified as primary or secondary. Primary GBM develops directly from glial cells. On the contrary, secondary GBM is when grade I glial tumors progress to become GBM.

[0514] GBM symptoms tend to come on quickly as the growing tumor puts pressure on the brain and cand destroy healthy brain tissue. Current known symptoms include, but are not limited to, blurred or double vision, headaches, loss of appetite, memory problems, mood or personality changes, muscle weakness or balance issues, nausea and vomiting, seizures, speech problems, changes in sensation, numbness or tingling.Therapeutic or Prophylactic Uses

[0515] The nucleic acid vaccines described herein may be used to prevent, alleviate and / or treat diseases, disorders and / or conditions caused by tumor-associated antigens. As a non-limiting example, the tumor-associated antigen may be, but is not limited to, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX, TTK, SPA 17, and EGFRvIII and a variant thereof.

[0516] The nucleic acid vaccines described herein may be used as prophylactic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of tumor-associated antigens for the prevention of cancer and / or tumors.

[0517] The nucleic acid vaccines described herein may be used as therapeutic agents where the nucleic acid vaccines are administered to a subject, and wherein the nucleic acid vaccine polynucleotide is translated in vivo to produce one or more proteins, peptides, fragments or variants thereof of tumor-associated antigens.

[0518] In some embodiments, provided are methods for prevention, alleviation and / or treatment of diseases, disorders and / or conditions caused by tumor-associated antigens, in a subject, by administering a nucleic acid vaccine comprising one or more polynucleotides encoding a tumor-associated antigen such as, but not limited to, MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX, TTK, SPA17, and EGFRvIII. The administration may be in combination with an anti-cancer agent or a small molecule compound described herein or known in the art.

[0519] In some embodiments, the nucleic acid vaccines described herein may be used to protect against and / or prevent the transmission of an emerging or engineered threat which may be known or unknown.I l l

[0520] In some embodiments, provided herein are methods of inducing translation of a polypeptide (e.g., one or more proteins, peptides, fragments or variants thereof of a tumor-associated antigen such as MAGE-A3, MAGE-A4, NY-ESO-1, PRAME, SSX, TTK, SPA17, and EGFRvIII) in a cell, tissue or organism using the nucleic acid polynucleotides described herein. The translated polypeptide may be used for the prevention, alleviation and / or treatment of cancer and / or tumors. Such translation can be in vitro, in vivo, ex vivo, or in culture. The cell, tissue or organism may be contacted with an effective amount of a composition or pharmaceutical composition containing the nucleic acid vaccine which includes a polynucleotide with at least one region encoding the polypeptide of interest described herein.

[0521] In some embodiments, provided herein is a sterile suspension containing a purified nucleic acid vaccine comprising a single-stranded 5’ capped (e.g., Cap 1 analog) mRNA which is formulated in a lipid nanoparticle (LNP). The nucleic acid vaccine may be produced by in vitro transcription. As a nonlimiting example, the sterile suspension comprises the nucleic acid vaccine encoding multiple tumor-associated antigens, and the mRNA sequence of the nucleic acid vaccine is provided as SEQ ID NO. 37. As a non-limiting example, the sterile suspension comprises the nucleic acid vaccine encodes multiple tumor-associated antigens, and the mRNA sequence of the nucleic acid vaccine is provided as SEQ ID NO. 36. As a non-limiting example, the sterile suspension comprises the nucleic acid vaccine encoding multiple tumor-associated antigens, where the mRNA sequence of the nucleic acid vaccine is provided as SEQ ID NO. 37 which is fully modified with N1 -methylpseudouridine. As a non-limiting example, the sterile suspension comprises the nucleic acid vaccine encoding multiple tumor-associated antigens, where the mRNA sequence of the nucleic acid vaccine is provided as SEQ ID NO. 36 which is fully modified with N1 -methylpseudouridine. The sterile suspension may be used for intramuscular administration.

[0522] In some embodiments, provided herein is a sterile suspension containing a purified nucleic acid vaccine comprising a single-stranded 5 ’capped mRNA comprising SEQ ID NO. 37 which is formulated in a lipid nanoparticle. The sterile suspension may be used for intramuscular administration.

[0523] In some embodiments, provided herein is a sterile suspension containing a purified nucleic acid vaccine comprising a single-stranded 5 ’capped mRNA comprising SEQ ID NO. 36 which is formulated in a lipid nanoparticle prepared using an ionizable lipid (Compound 201, 44% mol), a neutral lipid (DSPC, 10% mol), cholesterol (39% mol), a polymer-conjugated lipid (PEG2k-DMG, 2% mol) and a hydrophobic component (Squalene, 5% mol). The sterile suspension may be used for intramuscular administration.

[0524] In some embodiments, provided herein is a sterile suspension containing a purified nucleic acid vaccine comprising a single-stranded 5 ’capped mRNA comprising SEQ ID NO. 37 which is formulated in a lipid nanoparticle prepared using an ionizable lipid (Compound 201, 44% mol), a neutral lipid (DSPC, 10% mol), cholesterol (39% mol), a polymer-conjugated lipid (PEG2k-DMG, 2% mol) anda hydrophobic component (Squalene, 5% mol). The sterile suspension may be used for intramuscular administration.

[0525] In some embodiments, the nucleic acid vaccine is administered to a subject where antigen-presenting cells (APCs) to take up the mRNA of the nucleic acid vaccine, translate the mRNA into protein and present the resulting epitopes on human leukocyte antigen (HLA) molecules to stimulate an immune response against the encoded antigens of the nucleic acid vaccine. As anon-limiting example, the mRNA of the nucleic acid vaccine comprises SEQ ID NO. 37 and the mRNA is formulated in a lipid nanoparticle prepared using an ionizable lipid (Compound 201, 44% mol), a neutral lipid (DSPC, 10% mol), cholesterol (39% mol), a polymer-conjugated lipid (PEG2k-DMG, 2% mol) and a hydrophobic component (Squalene, 5% mol).

[0526] In some embodiments, the effective amount of the nucleic acid vaccine described herein provided to a cell, a tissue or a subject may be enough for the desired response and effectiveness. An effective amount of the composition containing the nucleic acid vaccine described herein is one that provides an induced or boosted immune response as a function of production in the cell of one or more proteins, polypeptides, peptides, fragments or variants thereof of tumor-associated antigen as compared to an untreated cell. Increased production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid vaccine), increased protein translation from the polynucleotide or altered innate immune response of the host cell.

[0527] Provided herein are directed to methods of inducing in vivo translation of one or more proteins, polypeptides, peptides, fragments or variants thereof of tumor-associated antigens in a mammalian subject in need thereof. An effective amount of a formulation comprising the nucleic acid vaccine containing at least one translatable region encoding the polypeptide described herein is administered to the subject using the delivery methods described herein. The polynucleotide is provided in an amount and under other conditions such that the polynucleotide is translated in the cell. The cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more rounds of nucleic acid vaccine administration.

[0528] The agents (e.g., compositions of nucleic acid vaccines and any additional moieties) can be administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery of both agents). The agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks. Generally, the agents may be concurrently bioavailable, e.g., detectable, in the subject. In some embodiments, the agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other embodiments, the agents may be delivered in separate unit dosages. The agents may be administered in any order, or as one or more preparations that includes two or more agents. In apreferred embodiment, at least one administration of one of the agents, e.g., the first agent, may be made within minutes, one, two, three, or four hours, or even within one or two days of the other agent, e.g., the second agent. In some embodiments, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least 25, 50, 75, 100, 200, 300, 400, or 500% greater than additive results.

[0529] In some embodiments, the compositions of nucleic acid vaccine described herein may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, the prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the prophylactic composition. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may 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, 2 hours, 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, 7 days, 8 days, 9 days, 10 days, 14 days, 21 days, 28 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 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.

[0530] In some embodiments, the nucleic acid vaccines may be formulated by the methods described herein. In one aspect, the formulation may comprise a nucleic acid vaccine or polynucleotide which can have a therapeutic and / or prophylactic effect on more than one disease, disorder or condition. As a nonlimiting example, the formulation may comprise polynucleotides encoding one or more proteins, polypeptide, peptides, fragments or variants thereof of tumor-associated antigens.

[0531] In some embodiments, the nucleic acid vaccines described herein, and compositions thereof, can be combined with adjuvants.

[0532] In some embodiments, the compositions of nucleic acid vaccines described herein can be administered to a subject prior to identification of the tumor-associated antigen or within 6 hours, 12 hours, 1 days, 2 days, 3 days or between 1 to 10 days after identification of the tumor-associated antigen. In some embodiments, the compositions of nucleic acid vaccines described herein can be first administered to a subject within 6 hours, 12 hours, 1 days, 2 days, 3 days or between 1 to 10 days after identification of the tumor-associated antigen and then repeat administration can occur within 6 hours, 12hours, 1 days, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, or between 1-2 weeks, 1-3 weeks, 1-4 weeks, 1-5 weeks, 1-6 weeks, 1-7 weeks, 1-8 weeks, 1-9 weeks, 1-10 weeks, 1-11 weeks, 1-12 weeks, 2-4 weeks, 2-5 weeks, 2-6 weeks, 2-7 weeks, 2-8 weeks, 2-9 weeks, 2-10 weeks, 2-11 weeks, 2-12 weeks, 1-2 months, 1-3 months, 1-4 months, 2-3 months, 2-4 months, 2-5 months, 2-6 months, 2-7 months, 3-6 months, 3-9 months, 6-9 months, 6-12 months, 9-12 months, yearly, biannually, triennially or more after the first administration. Both administrations may be intramuscular administrations. Both administrations may be intramuscular administrations. Alter...

Claims

1. CLAIMS1. A polynucleotide comprising a sequence encoding at least one region from each of five tumor-associated antigens, or a fragment, peptide or variant thereof, wherein said tumor- associated antigens are selected from the group consisting of MAGE-A3, MAGE-A4, NY- ESO-1, PRAME, SSX2, TTK and SPA17.

2. The polynucleotide of claim 1, wherein the polynucleotide comprises a first sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 21, a second sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO.22, a third sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 23, a fourth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 24, a fifth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 25, a sixth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 26, a seventh sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 27, an eighth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 28, a ninth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 29, a tenth sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 32, and an eleventh sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 33.

3. The polynucleotide of claim 2, wherein the polynucleotide further comprises a twelfth sequence region and a thirteenth sequence region, wherein the twelfth sequence region has a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 30 and the thirteenth sequence region has a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 31.

4. The polynucleotide of claim 3, wherein the polynucleotide further comprises at least one fourteenth sequence region, wherein the at least one fourteenth sequence region comprising the nucleic acid sequence of SEQ ID NO. 103-114 or 128-139.

5. The polynucleotide of claim 4, wherein the polynucleotide further comprises at least one fifteenth sequence region, wherein the at least one fifteenth sequence region comprising the nucleic acid sequence of SEQ ID NO. 142, 145, or 148.

6. The polynucleotide of claim 5, wherein the polynucleotide further comprise a sixteenth sequence region, said sixteenth sequence region comprising the nucleic acid sequence of SEQ ID NO. 89.

7. The polynucleotide of claim 2 or 3, comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO. 34, 35, 38, 39, 46, 47, 50, or 51, preferably comprising a nucleic acid sequence consisting of SEQ ID NO. 34, 35, 38, 39, 46, 47, 50, or 51.

8. The polynucleotide of claim 2, comprising a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO. 42 or 43, preferably comprising a nucleic acid sequence consisting of SEQ ID NO. 42 or 43.

9. A polynucleotide comprising a sequence encoding epidermal growth factor receptor variant III (EGFRvIII), or a fragment, peptide or variant thereof, wherein the polynucleotide comprises a first sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO. 166, 167, 168, 169 or 170.

10. The polynucleotide of claim 9, wherein said first sequence region consists of SEQ ID NO.166, 167, 168, 169, or 170.

11. The polynucleotide of any one of claims 1-10, wherein at least 50% of the polynucleotide sequence is codon optimized.

12. The polynucleotide of any one of claims 1 to 11, wherein the polynucleotide is a RNA, or an mRNA.

13. The polynucleotide of claim 12, wherein the mRNA comprises a 5’UTR and a 3’UTR, wherein said 5’UTR comprises SEQ ID NO. 150 and said 3’UTR comprises SEQ ID NO. 152, 154, 156 or 158.

14. The polynucleotide of claim 13, wherein at least one, or preferably all, uracil nucleosides are modified to be N1 -methylpseudouridine.

15. A polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO. 36, 37, 40, 41, 44, 45, 48, 49, 52, 53, 171, 172, 174, 175, 177, 178, 180, 181, 183, and 184.

16. A nucleic acid vaccine comprising the polynucleotide of any one of claims 1-15.

17. The nucleic acid vaccine of claim 16, formulated in a lipid nanoparticle (LNP), wherein the LNP preferably comprises (a) from about 40 to about 100 mol% of an ionizable lipid, (b) from about 0 to about 10 mol% of a neutral lipid, (c) from about 0 to about 50mol% of a helper lipid, (d) from about from 0 to about 5 mol% of a polymer-conjugated lipid, and (e) from 0 to about 5 mol% of a hydrophobic component, wherein the mol% are based on the total lipids present in the nanoparticle.

18. The nucleic acid vaccine of claim 17, wherein the ionizable lipid is Compound 201.

19. A pharmaceutical composition comprising the nucleic acid vaccine of any one of claims 16 to 18 and a pharmaceutically acceptable excipient.

20. A method of inducing an immune response in a subject comprising administering the nucleic acid vaccine of any one of claims 16 to 18 or the pharmaceutical composition of claim 19 to the subject.

21. The nucleic acid vaccine of any one of claims 16 to 18 for use in induction of an immune response in a subject in need thereof.

22. The method of claim 20 or the nucleic acid vaccine for use of claim 21, wherein the immune response comprises a T-cell response.

23. A method of treating cancer in a subject comprising administering the nucleic acid vaccine of claims 16 to 18 to the subject, wherein the nucleic acid vaccine induces an immune response against cancer cells.

24. A nucleic acid vaccine of any one of claims 16 to 18 for use in the treatment of cancer in a subject in need thereof, wherein the use of the nucleic acid vaccine induces an immune response against cancer cells.

25. The method of claim 23 or the nucleic acid vaccine for use of claim 24, wherein the cancer is a solid tumor, basal cell carcinomas, bladder cancer, brain cancer, cholangiocarcinoma, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, female reproductive system-associated carcinomas, endometrial carcinomas, uterine serous carcinomas, gastric cancer, glioblastoma, glioma, head and neck cancer, head and neck squamous cell carcinoma (HNSCC), hepatocellular cancer (HCC), kidney cancer, leukemia, acute myeloid leukemia (AML), lymphoma, diffuse large B cell lymphoma (DLBCL), lung cancer, non-small cell lung cancer (NSCLC), melanoma, advanced melanoma, uveal melanoma, metastatic melanoma, cutaneous melanoma, multiple myeloma, mesothelioma, nervous system- associated tumors, neuroblastoma, ovarian cancer, ovarian clear cell carcinomas, pancreatic cancer, pancreatic adenocarcinoma, prostate cancer, renal cell carcinoma, sarcoma, synovial sarcoma, soft tissue sarcoma, osteosarcoma, liposarcoma, or thymic carcinomas.

26. The method of claim 23 or the nucleic acid vaccine for use of claim 24, wherein the nucleic acid vaccine is administered six times over a nine-week period in the subject.

27. The method of any one of claims 23, 25 and 26 or the nucleic acid vaccine for use of any one of claims 24, 25 and 26, wherein the cancer is:i) a solid tumor;ii) a head and neck cancer; orii) a glioblastoma, wherein the glioblastoma is optionally a GBM with leptomeningeal spread.

28. The method of claim 23 or the nucleic acid vaccine for use of claim 24, wherein the treatment of the cancer comprises:i) a reduction in tumor volume;ii) a reduction in tumor burden; and / oriii) an alteration of the phenotype of the tumor environment.

29. The method of claim 23 or the nucleic acid vaccine for use of claim 24, wherein the administration or the use of the nucleic acid vaccine results in increased CD8 T cell response as compared to a control.

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