Lipid nanoparticles for delivery of nucleic acids

Abstract

Provided are compositions comprising lipid nanoparticles comprising a galactosylceramide, which can be used to deliver nucleic acids, such as ribonucleic acids (RNA). In some embodiments, the lipid nanoparticles comprises an alpha-galactosylceramide (α-GalCer). In some aspects, the α- GalCer helps enhance immune responses in a subject to which the lipid nanoparticles are delivered, and can deliver the payload to particular location in the body, including the tumor and tumor- draining lymph nodes. Also provided are compositions comprising the lipid nanoparticles and methods of using the lipid nanoparticles, including therapeutic and prophylactic methods.

Description

LIPID NANOPARTICLES FOR DELIVERY OF NUCLEIC ACIDSCross-Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 505,985, filed June 2. 2023, entitled "LIPID NANOPARTICLES FOR DELIVERY OF NUCLEIC ACIDS”, which is hereby incorporated herein by reference in its entirety.Field

[0002] The present disclosure relates to compositions comprising lipid nanoparticles comprising a galactosylceramide, which can be used to deliver nucleic acids, such as ribonucleic acids (RNA). In some embodiments, the lipid nanoparticles comprises an alphagalactosylceramide (a-GalCer). In some aspects, the a-GalCer helps enhance immune responses in a subject to which the lipid nanoparticles are delivered, and can deliver the pay load to particular location in the body, including the tumor and tumor-draining lymph nodes. Also provided are compositions comprising the lipid nanoparticles and methods of using the lipid nanoparticles, including therapeutic and prophylactic methods.Background

[0003] Lipid nanoparticles have been used as pharmaceutical drug delivery systems, to deliver a variety of payload, including drugs. However, challenges exist in efficient and targeted delivery of the payload to the subject using lipid nanoparticles. There remains a need for efficient method for lipid nanoparticle-based drug delivery’, for targeted delivery of the pay load to particular locations in the subject’s body, and in some cases to enhance immunogenicity. Provided are embodiments that meet such needs.Summary’

[0004] Provided herein are nanoparticles, for example for delivery of nucleic acids. Provided are nanoparticles comprising N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA); l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and a galactosylceramide. In some of any embodiments, the galactosylceramide is alpha-galactosylceramide (a-GalCer).

[0005] In some of any embodiments, the nanoparticle further comprises a nucleic acid. In some of any embodiments, the nucleic acid comprises a ribonucleic acid (RNA). In some of any embodiments, the nucleic acid comprises a circular RNA. In some of any embodiments, the nucleic acid comprises a linear RNA. In some of any embodiments, the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2',3'-cyclic phosphate at the 3' terminus. In some of any embodiments, the nucleic acid comprises a linear messenger RNA (mRNA). In some of any embodiments, the nucleic acid comprises a micro RNA (miRNA). In some of any embodiments, the nucleic acid comprises a small inhibitory RNA (siRNA). In some of any embodiments, the nucleic acid comprises a guide RNA (gRNA).

[0006] In some of any embodiments, the nanoparticle forms a lipoplex (LPX).

[0007] In some of any embodiments, the ratio of DOTMA:DOPE is between about 5: 1 and about 1 :5. In some of any embodiments, the ratio of DOTMA:DOPE is between about 3: 1 and about 1 :3. In some of any embodiments, the ratio of DOTMA:DOPE is about 2: 1.

[0008] In some of any embodiments, the proportion of a-GalCer is between about 0. 1 % and about 5% molar percentage. In some of any embodiments, the proportion of a-GalCer is between about 0.5% and about 2% molar percentage. In some of any embodiments, the proportion of a- GalCer is about 0.5% molar percentage. In some of any embodiments, the proportion of a-GalCer is about 1 % molar percentage. In some of any embodiments, the proportion of a-GalCer is about 2% molar percentage.

[0009] In some of any embodiments, the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is between about 1:2 to about 2: 1. In some of any embodiments, the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is about 1.3:2.

[0010] In some of any embodiments, the nucleic acid comprises a nucleic acid sequence encoding one or more exogenous molecules.

[0011] In some of any embodiments, the one or more exogenous molecules is selected from among a vaccine epitope, a cancer epitope, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

[0012] In some of any embodiments, the one or more exogenous molecule comprises a vaccine epitope. In some of any embodiments, the vaccine epitope comprises an infectious disease vaccine epitope, optionally a viral vaccine epitope. In some of any embodiments, the vaccine epitope comprises a cancer epitope. In some of any embodiments, the cancer epitope isa cancer neoantigen epitope. In some of any embodiments, the cancer neoantigen epitope comprises a cancer epitope selected from among ME-1, M26, Gludl, Mtchl, M18, and E2f8.

[0013] In some of any embodiments, the one or more exogenous molecules comprises a sequence-specific nuclease. In some of any embodiments, the sequence-specific nuclease is a Cas nuclease. In some of any embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a CasI2 nuclease, or a Casl3 nuclease.

[0014] In some of any embodiments, the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

[0015] In some of any embodiments, the one or more exogenous molecule comprises an immunomodulatory polypeptide. In some of any embodiments, the immunomodulatory polypeptide comprises a cytokine.

[0016] In some of any embodiments, the one or more exogenous molecules comprises a transcription factor.

[0017] In some of any embodiments, the one or more exogenous molecules comprises a reporter molecule. In some of any embodiments, the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

[0018] In some of any embodiments, when the nanoparticle is administered to a subject, an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle.

[0019] In some of any embodiments, the immune response comprises an increase in cytokine production. In some of any embodiments, the cytokine is selected from among one or more of: IFN- y, IL-2, IL-6, and IL- 12.

[0020] In some of any embodiments, the immune response comprises an increase in costimulatory molecule expression on dendritic cells. In some of any embodiments, the costimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.

[0021] In some of any embodiments, the immune response comprises activation of immune cells. In some of any embodiments, the immune cell is selected from among one or more of: NKT. NK, CD4+ T, and CD8+ T cells.

[0022] In some of any embodiments, the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity of tumor- specific T cells, and increase in migration of tumor-specific T cells.

[0023] In some of any embodiments, the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.

[0024] In some of any embodiments, when administered to a subject, the nanoparticle is localized to the spleen. In some of any embodiments, when administered to a subject, the nanoparticle is localized to a tumor. In some of any embodiments, when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node. In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen.

[0025] In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor. In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumordraining lymph node. In some of any embodiments, when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor growth, or increasing survival of the subject.

[0026] Provided herein are methods for generating a nanoparticle. In some of any embodiments, the methods involve generating a lipid mixture of N-[l-(2,3-dioleyloxy)propyl]- N,N,N-trimethylammonium chloride (DOTMA) and l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE); and incorporating an a galactosylceramide to the mixture. In some of any embodiments, the galactosylceramide is alpha-galactosylceramide (a-GalCer).

[0027] In some of any embodiments. The method forms a lipoplex (LPX).

[0028] In some of any embodiments, the ratio of DOTMA: DOPE is bet een about 5: 1 and about 1 :5. In some of any embodiments, the ratio of DOTMA: DOPE is between about 3: 1 and about 1 :3. In some of any embodiments, the ratio of DOTMA:DOPE is about 2: 1.

[0029] In some of any embodiments, the proportion of a-GalCer is between about 0.1% and about 5% molar percentage. In some of any embodiments, the proportion of a-GalCer is between about 0.5% and about 2% molar percentage. In some of any embodiments, the proportion of a- GalCer is about 0.5% molar percentage. In some of any embodiments, the proportion of a-GalCer is about 1% molar percentage. In some of any embodiments, the proportion of a-GalCer is about 2% molar percentage.

[0030] In some of any embodiments, the methods also involve mixing the nanoparticle with a nucleic acid.

[0031] In some of any embodiments, the nucleic acid comprises a ribonucleic acid (RNA). In some of any embodiments, the nucleic acid comprises a circular RNA. In some of any embodiments, the nucleic acid comprises a linear RNA. In some of any embodiments, the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2', 3'- cyclic phosphate at the 3' terminus. In some of any embodiments, the nucleic acid comprises a linear messenger RNA (mRNA). In some of any embodiments, the nucleic acid comprises a micro RNA (miRNA). In some of any embodiments, the nucleic acid comprises a small inhibitory RNA (siRNA). In some of any embodiments, the nucleic acid comprises a guide RNA (gRNA).

[0032] In some of any embodiments, the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is between about 1:2 to about 2: 1. In some of any embodiments, the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is about 1.3:2.

[0033] Also provided are nanoparticles generated by any of the methods described herein.

[0034] Also provided are compositions comprising any of the nanoparticles described herein. In some of any embodiments, the composition is a pharmaceutical composition. In some of any embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0035] Also provided are methods of vaccinating a subject. In some of any embodiments, the methods involve administering any of the nanoparticles described herein, or any of the compositions described herein to a subject.

[0036] Also provided are methods for enhancing an immune response to a vaccine epitope in a subject. In some of any embodiments, the methods involve administering any of the nanoparticles described herein, or any of the compositions described herein to a subject.

[0037] Also provided are methods of treating a disease or disorder in a subject. In some of any embodiments, the methods involve administering any of the nanoparticles described herein, or any of the compositions described herein to a subject.

[0038] In some of any embodiments, an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle or the composition.

[0039] In some of any embodiments, the immune response comprises an increase in cytokine production. In some of any embodiments, the cytokine comprises one or more of: IFN- y, IL-2. IL-6, and IL-12. In some of any embodiments, the cytokine comprises IFN-y.

[0040] In some of any embodiments, the immune response comprises an increase in costimulatory molecule expression on dendritic cells. In some of any embodiments, the immune response comprises an increase in co-stimulatory molecule expression on MHC-II+CD11b- CD1 lc+ dendritic cells. In some of any embodiments, the immune response comprises an increase in co- stimulatory molecule expression on MHC-II+ CD1 Ib+CDl lc+ dendritic cells. In some of any embodiments, the co-stimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.

[0041] In some of any embodiments, the immune response comprises activation of an immune cell. In some of any embodiments, the immune cell is selected from among one or more of: NKT. NK, CD4+ T, and CD8+ T cells.

[0042] In some of any embodiments, the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity of tumor-specific T cells, and increase in migration of tumor-specific T cells.

[0043] In some of any embodiments, the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.

[0044] In some of any embodiments, when administered to a subject, the nanoparticle is localized to the spleen. In some of any embodiments, when administered to a subject, the nanoparticle is localized to a tumor. In some of any embodiments, when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node.

[0045] In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen. In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor. In some of any embodiments, when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor-draining lymph node.

[0046] In some of any embodiments, when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor growth, or increasing survival of the subject.

[0047] In some of any embodiments, the method prevents or reduces the severity of a disease or disorder associated with the vaccine epitope in the subject.

[0048] In some of any embodiments, the disease or disorder is an infectious disease, optionally a viral infection.

[0049] In some of any embodiments, the disease or disorder is a cancer.

[0050] In some of any embodiments, the administration is by intravenous administration.

[0051] Also provided are any of the nanoparticles described herein, or any of the compositions described herein for use in vaccinating a subject, wherein the nanoparticle, or the composition is administered to the subject.

[0052] Also provided are any of the nanoparticles described herein, or any of the compositions described herein for use in enhancing an immune response to a vaccine epitope in a subject, wherein the nanoparticle, or the composition is administered to the subject.

[0053] Also provided are any of the nanoparticles described herein, or any of the compositions described herein for use in treating a disease or disorder in a subject, wherein the nanoparticle, or the composition is administered to the subject.

[0054] Also provided are uses of any of the nanoparticles described herein, or any of the compositions described herein in the manufacture of a medicament for vaccinating a subject, wherein the medicament is administered to the subject.

[0055] Also provided are uses of any of the nanoparticles described herein, or any of the compositions described herein in the manufacture of a medicament for enhancing an immune response to a vaccine epitope in a subject, wherein the medicament is administered to the subject.

[0056] Also provided are uses of any of the nanoparticles described herein, or any of the compositions described herein in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.Brief Description of the Drawings

[0057] FIGS. 1A-1D shows characteristics and physical properties of exemplary nanoparticles provided herein, including the LPX / a-GalCer / circular RNA nanoparticles. FIG. 1A shows a schematic representing an exemplary LPX / a-GalCer / circular RNA nanoparticle composed of DOTMA, DOPE, and a-GalCer with circular RNA in the center. FIG. IB shows average particle size for compositions of LPX / a-GalCer / circular RNA nanoparticles with either no a-GalCer, or 0.5% a-GalCer, 1% a-GalCer, or 2% a-GalCer. FIG. 1C shows the poly dispersity index (PDI) for compositions of LPX / a-GalCer / circular RNA nanoparticles with either no a-GalCer, or 0.5 % a- GalCer, 1% a-GalCer, or 2% a-GalCer. FIG. ID shows thezeta-potential for compositions of LPX / a-GalCer / circular RNA nanoparticles with either no a- GalCer, or 0.5% a-GalCer, 1% a- GalCer, or 2% a-GalCer.

[0058] FIGS. 2A-2B shows in vivo localization of LPX / a-GalCer / circular RNA with the RNA encoding a firefly luciferase in mice. FIG. 2A shows bioluminescence imaging results of mice 6 hours after the injection of LPX / a-GalCer / circular RNA with the RNA encoding firefly luciferase, with firefly luciferase activity appearing in the spleen region. FIG. 2B shows organs from mice at 24 and 48 hours after injection with LPX / a-GalCer / circular RNA encoding firefly luciferase where firefly luciferase activity is predominantly seen in the spleen.

[0059] FIGS. 3A-3H shows mice injected with LPX / a-GalCer / circular RNA with the RNA encoding eGFP. 6 hours after injection splenocytes were isolated and the mean fluorescence intensity (MFI) was measured by flow cytometry to determine cells that LPX / a- GalCer / circular RNA localized to. FIG. 3A shows a schematic of the experiment demonstrating LPX / a-GalCer / circular RNA was injected through the tail vein and splenocytes were isolated 6 hours after injection. FIG. 3B shows the mean fluorescence intensity (MFI) of eGFP in dendritic (CD1 lc+, MHCII+) cells between control mice and mice injected with LPX / a- GalCer / circular RNA. FIG. 3C shows the MFI of eGFP in macrophages (CD1 lb+, Ly6G" cells) between control mice and mice injected with LPX / a-GalCer / circular RNA. FIG. 3D shows the MFI of eGFP in neutrophils (CD1 lb+, Ly6G+cells) cells between control mice and mice injected with LPX / a-GalCer / circular RNA. FIG. 3E shows the MFI of eGFP in CD4 T cells (CD3+, CD4+cells) between control mice and mice injected with LPX / a-GalCer / circular RNA. FIG. 3F shows the MFI of eGFP in CD8+T cells (CD3+, CD8+cells) between control mice and mice injected with LPX / a-GalCer / circular RNA. FIG. 3G shows the MFI of eGFP in B cells (B220+cells) between control mice and mice injected with LPX / a-GalCer / circular RNA. FIG. 3H shows the MFI of eGFP in natural killer (NK) cells (NK1.1+cells) between control mice and mice injected with LPX / a-GalCer / circular RNA.

[0060] FIG. 4 show s a schematic of an exemplary process of how' after LPX / a-GalCer / circular RNA nanoparticles are injected and delivered to dendritic cells (DCs), a- GalCer may be presented on the cell surface by CD Id, which may be recognized by T cell receptors on natural killer T cells (NKT) cells which may lead NKT cells to become activated and secrete various cytokine including interferon gamma (IFN-y), which subsequently activates various immune cells including DCs, macrophages, T cells, and NK cells.

[0061] FIGS. 5A-5E show the results of a multiplex cytokine assay on mouse serum 6 hours after injection with LPX / a-GalCer / circular RNA for cytokines associated with NK cell activation. FIG. 5A shows a schematic of an exemplary process for the experiment demonstrating that mice are injected with LPX / circular RNA, LPX / 0.5% a-GalCer / circular RNA, LPX / 1% a- GalCer / circular RNA, or LPX / 2% a-GalCer / circular RNA and cytokine assays are done 6 hours after injection. FIG. 5B shows IFN-y concentration in mouse serum 6 hours after injection with LPX / circular RNA, LPX / 0.5% a-GalCer / circular RNA, LPX / 1% a- GalCer / circular RNA, or LPX / 2% a-GalCer / circular RNA. FIG. 5C shows IL-2 concentrations in mouse serum 6 hours after injection with LPX / circular RNA. LPX / 0.5% a-GalCer / circular RNA, LPX / 1% a-GalCer / circular RNA. or LPX / 2% a-GalCer / circular RNA. FIG. 5D shows IL-6 concentration in mouse serum 6 hours after injection with LPX / circular RNA, LPX / 0.5% a-GalCer / circular RNA, LPX / 1% a- GalCer / circular RNA, or LPX / 2% a-GalCer / circular RNA. FIG. 5E shows IL-12 concentration in mouse serum 6 hours after injection with LPX / circular RNA, LPX / 0.5% a-GalCer / circular RNA, LPX / 1% a-GalCer / circular RNA, or LPX / 2% a- GalCer / circular RNA.

[0062] FIGS. 6A-6K shows results from flow cytometry of cell types after inj ection of either LPX / circular RNA or LPX / 2% a-GalCer / circular RNA by either intramuscular (IM) or intravascular (IV) injection. FIG. 6A shows a schematic of the experiment where mice are injected with either LPX / circular RNA or LPX / 2% a-GalCer / circular RNA by either IM or IV injections with flow cytometry being performed 3 days after injection on splenocytes. FIG. 6B shows the MFI for CD40 in dendritic cells (DC) that are MHCII+, CDllb", and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a- GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a-GalCer / circular RNA. FIG. 6C shows the MFI for CD80 in DCs that are MHCIL, CD1 lb‘, and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a-GalCer / circular RNA. FIG. 6D shows the MFI for CD86 in DCs that are MHCIL, CD1 lb", and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA. IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA.

[0063] FIG. 6E shows the MFI for CD40 in DCs that are MHCIL, CD1 lb+, and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a- GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a-GalCer / circular RNA.FIG. 6F shows the MFI for CD80 in DCs that are MHCII+. CD1 lb+, and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a-GalCer / circular RNA. FIG. 6G shows the MFI for CD86 in DCs that are MHCII+, CD1 lb+, and CD1 lc+from spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA.

[0064] FIG. 6H shows the percent of cells positive for CD69 in natural killer T (NKT) cells (CD45+, CD3+, CD49b+cells) in spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA. FIG. 61 shows the percent of cells positive for CD69 in natural killer (NK) cells (CD45+, CD3‘, CD49b+cells) in spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA. FIG. 6J shows the percent of cells positive for CD69 in CD4 T cells (CD45+, CD3+, CD4+cells) in spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA. FIG. 6K shows the percent of cells positive for CD69 in CD8 T cells (CD45+, CD3+, CD8+cells) in spleens of mice that are control, IM with LPX / circular RNA, IM with LPX / 2% a-GalCer / circular RNA, IV with LPX / circular RNA, or IV with LPX / 2% a- GalCer / circular RNA.

[0065] FIGS. 7A-7G show an experiment where mice were injected with CT26 cells to model a tumor followed by injection with LPX / a-GalCer / circular RNA encoding firefly luciferase wither by IM or IV injection before the organs were harvested to measure luciferase activity in different location in the body. FIG. 7A shows a schematic of the experimental design showing that CT26 cells are injected and after injection with CT26 cells, 7 days later mice are either used as controls, injected IM with LPX / a-GalCer / circular RNA encoding firefly luciferase, or injected IV with LPX / a-GalCer / circular RNA encoding firefly luciferase. Organs are then harvested the next day, which is 8 days after the mice were injected with CT26. FIG. 7B shows a schematic of the organs harvested to detect luciferase activity in. with an axillary lymph node (LN), spleen, mesenteric LNs, Inguinal LNs, and the tumor harvested. FIG. 7C shows the luciferase activity in the spleen of mice that are controls, IM with LPX / a- GalCer / circular RNA encoding firefly luciferase or IV with LPX / a-GalCer / circular RNA encoding firefly luciferase. FIG. 7D shows the luciferase activity in the tumor of mice that arecontrols, IM with LPX / a-GalCer / circular RNA encoding firefly luciferase or IV with LPX / a- GalCer / circular RNA encoding firefly luciferase. FIG. 7E shows the luciferase activity in the inguinal LN of mice that are controls, IM with LPX / a-GalCer / circular RNA encoding firefly luciferase or IV with LPX / a-GalCer / circular RNA encoding firefly luciferase. FIG. 7F shows the luciferase activi ty in the axillary LN of mice that are controls. IM with LPX / a- GalCer / circular RNA encoding firefly luciferase or IV with LPX / a-GalCer / circular RNA encoding firefly luciferase. FIG. 7G shows the luciferase activity in the mesenteric LN of mice that are controls, IM with LPX / a-GalCer / circular RNA encoding firefly luciferase or IV with LPX / a-GalCer / circular RNA encoding firefly luciferase.

[0066] FIGS. 8A-8C shows an experiment measuring the anti-cancer effects of LPX / a- GalCer / circular RNA encoding neoantigens alone, LPX / circular RNA encoding various neoantigens alone, PD-1 inhibitor monotherapy, LPX / a-GalCer / circular RNA encoding neoantigens administered with PD-1 inhibitor, or LPX / circular RNA encoding various neoantigens administered with PD-1 inhibitor. FIG. 8A shows a schematic of the experiment with CT26 cells injected at the start and then IV with LPX / a-GalCer / circular RNA encoding neoantigens alone, LPX / circular RNA encoding various neoantigens alone, PD-1 inhibitor monotherapy, LPX / a-GalCer / circular RNA encoding neoantigens administered with PD-1 inhibitor, or LPX / circular RNA encoding various neoantigens administered with PD-1 inhibitor after which mice were assessed for tumor growth and survival. FIG. 8B, shows the tumor growth across mice treated with LPX / a-GalCer / circular RNA encoding neoantigens alone, LPX / circular RNA encoding various neoantigens alone, PD-1 inhibitor monotherapy, LPX / a- GalCer / circular RNA encoding neoantigens administered with PD-1 inhibitor, or LPX / circular RNA encoding various neoantigens administered with PD-1 inhibitor. FIG. 8C, shows the survival rate of mice treated with LPX / a-GalCer / circular RNA encoding neoantigens alone, LPX / circular RNA encoding various neoantigens alone, PD-1 inhibitor monotherapy, LPX / a- GalCer / circular RNA encoding neoantigens administered with PD-1 inhibitor, or LPX / circular RNA encoding various neoantigens administered with PD-1 inhibitor.

[0067] FIGS. 9A-9D shows LPX / a-Galcer nanoparticle targets eDCs and pDCs in the spleen. FIG. 9A shows a schematic of the experiment with LPX-aGalcer nanoparticle (LPXa, 100 pl) or LPX encapsulating mRNA encoding eGFP (10 pg) injected into the vein of mice at the start then FC of the splenocytes at 6 hour (PBS was used as a control). FIG. 9B shows EGFP-positive cells being observed by fluorescence microscopy when splenocytes wereisolated from the spleen 6 hours after injection. FIG. 9C shows EGFP+cells among conventional dendritic cells (eDC, Lin‘CD45+CDl lchlMHC-IIhl) cells by flowcytometry of splenocytes isolated from mice intravenously injected with LPXa, LPX, or PBS, respectively. FIG. 9D shows the analyzed % of EGFP+cells among gated cells as eDCs (Lin" CD45+CD1 lchlMHC-IIhl), plasmacytoid dendritic cells (pDCs, Lin’CD45+CDl lcloMHC- IIhlCD137hl), macrophages (Lin'CD45+F4 / 80+CDl lb+), neutrophils (Lin CD45 CDl lb+Ly6G+), B cells (CD3 CD19 ), CD4+T, CD8+T cells.Detailed Description

[0068] Provided herein are lipid nanoparticles comprising a galactosylceramide. In some aspects, the provided nanoparticles comprise N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA); l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and a galactosylceramide. In some aspects, the galactosylceramide is an alpha- galactosylceramide (a-GalCer). In some aspects, the provided nanoparticles include a nucleic acid payload. In some aspects, the provided nanoparticles can be used to deliver a nucleic acid pay load. In some aspects, the a-GalCer component of the nanoparticle stimulates an immune response. In some aspects, the a-GalCer component also allows selective targeting of the pay load contained in the nanoparticle to particular regions or areas of a subject, after administration of the nanoparticle or composition. In some aspects, also provided are methods and uses, including prophylactic and therapeutic methods and uses, such as vaccination methods, that employ the provided nanoparticles. Also provided are methods and uses of the provided nanoparticles such as in therapeutic and prophylactic uses, such as in treating a disease or disorder or vaccination of a subject. Also provided are methods for producing the nanoparticles.

[0069] In some aspects, the provided embodiments can result in improved immune response against the delivered payload, such as a nucleic acid encoding a vaccine epitope, increase in anti-vaccine epitope immune response, improved vaccination and improved therapeutic outcome (e.g., anti-cancer therapy). In some contexts, the provided embodiments, including combination therapy, result in increased efficiency and selective targeting of delivery of the payload, such as nucleic acid pay load, can lead to improved induction of an immune response and therapeutic effect, including anti-cancer immune response.

[0070] In some aspects, the provided embodiments are based on an observation as described herein that exemplary lipid nanoparticles comprising a-GalCer resulted in substantial induction of immune stimulator}' cytokines such as IFN-y, IL-2, IL-6, and IL-12, upregulation of co-stimulatory molecules such as CD40, CD80, and CD86 on dendritic cells, and activation of NKT, NK. CD4+ T. and CD8+ T cells, when administered intravenously. In addition, the exemplary hpid nanoparticles comprising a-GalCer also unexpectedly led to selective targeting and delivery of the nucleic acid payload to the spleen, tumor and tumor-draining lymph nodes, but not to non-tumor-draining lymph nodes, when administered intravenously. The results described herein indicate that the exemplary lipid nanoparticles comprising a-GalCer can activate not only systemic but also tumor microenvironmental immunity. The surprising ability of the exemplary nanoparticles to target the tumor and tumor-draining lymph nodes support that the provided nanoparticles and compositions can be used for targeted delivery of the payload. The results also support that the provided nanoparticles and compositions can specifically enhance anti-tumor immune responses, as tumor- draining lymph nodes play a central role in the generation and proliferation of tumor-specific T cells, increase in tumor-specific cytolytic activity and migration. The provided nanoparticles and compositions have been observed to effectively control tumor grow th by promoting anti-cancer immunity7.

[0071] As exemplified herein, administration of exemplary lipid nanoparticles comprising a- GalCer and nucleic acids encoding cancer neoantigen epitopes, efficiently controlled tumor growth and improved survival rate at an extent similar to a PD- 1 inhibitor therapy. In comparison, lipid nanoparticles with the same nucleic acids but without a-GalCer, alone did not result in substantial inhibition of tumor growth or improved survival. The provided embodiments were observed to improve anti-cancer therapy, by reducing the tumor size and increasing the survival of subjects administered exemplary lipid nanoparticles comprising a-GalCer and nucleic acids encoding cancer neoantigen epitopes. As described herein, substantial improvements in inhibiting tumor growth and survival w ere observed. The results described herein support the various advantages of the provided nanoparticles and compositions.

[0072] In some aspects, the provided embodiments offer various advantages and improvements in producing immune responses, such as anti-tumor immune responses or immune responses against vaccines, particularly to elicit epitope-specific T cell activity, for therapy and / or vaccination. In some aspects, the provided embodiments are based on theseobservations that show substantially improved therapeutic outcome, particularly those that have been difficult using existing approaches, such as anti-cancer vaccination. The provided nanoparticles, such as a lipoplex (LPX) nanoparticles comprising a-GalCer, and compositions, can be employed in various therapeutic and prophylactic uses, including for example infectious disease vaccines, cancer vaccines, immunotherapy, autoantigen vaccines, gene therapy, gene editing and others.

[0073] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0074] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. LIPID NANOPARTICLES

[0075] Provided herein are nanoparticles comprising N-[l-(2.3-dioleyloxy)propyl]- N,N,N- trimethylammonium chloride (DOTMA); 1.2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE); and a galactosylceramide. In some aspects, the galactosylceramide is an alpha-galactosylceramide (a-GalCer). In some aspects, the provided nanoparticles include a nucleic acid payload, or can be used to deliver a nucleic acid payload. Also provided are methods and uses of the provided nanoparticles such as in therapeutic and prophylactic uses, such as in treating a disease or disorder or vaccination of a subject. The methods and uses provided herein may be applicable for treating various patients with different diseases, developing therapeutics, and / or developing vaccines against various diseases, including infectious diseases and cancers. Also provided are methods for producing the nanoparticles.A. Galactosylceramide

[0076] In some aspects, the nanoparticles provided herein comprise a galactosylceramide as a component. In some aspects, the galactosylceramide is alpha- galactosylceramide (a-GalCer, also known as KRN7000). a-GalCer is an a-galactosylatedsphingolipid comprising an a-linked sugar and lipid moieties (Morita et al., (1995) Journal of Medicinal Chemistry. 38 (12): 2176-2187). It is naturally occurring in bacteria and marine sponges (Agelas mauritianus) or can be synthetically produced. In some aspects, a-GalCer was found to regulate metabolism and the immune system within humans, specifically a natural ligand to CD Id and once bound to the receptor can activate natural killer T (NKT) cells, a- GalCer is a potent activator of invariant NKT cells, and is a CDld antigen. In some aspects, the invariant T cell receptor of the iNKT cell is able to bind the CDld:gly colipid complex leading to iNKT cell activation. In some contexts, activation of NKT cells leads to the increased release of cytokines and cytotoxicity mediated by the cells. NKT cells release immunomodulatory cytokines and are important for modulating and regulating the immune response. This can include coordinating immune cells in anti-tumor immunity and activation of NKT cells can lead to a decrease in tumor burden, but they also play a role in autoimmune diseases. Many autoimmune diseases have been found to be exacerbated through NKT-cell deficiency and activation of NKT cells with glycolipid antigens, like a-GalCer, have been found to alleviate protects mice against autoimmunity.

[0077] In some aspects, a-GalCer has adjuvant activity and can stimulate a strong immune response against a peptide vaccine epitope that is administered in conjunction with the a-GalCer. In some cases, the CDld:glycolipid:T cell receptor (TCR) interaction activates the iNKT cells, which in turn activates dendritic cells (DCs). In some aspects, such activation can lead to production and release of cytokines and stimulates the DCs to activate epitope-specific T cell response. In some embodiments, the a-GalCer is presented to NKT cells. In some embodiments, the a-GalCer binds to and is presented on CDld forming a CDld / a-GalCer complex on a cell expressing CDld. In some embodiments, the CDld / a-GalCer complex binds T cell receptors (TCR) on NKT cells. In some embodiments, CD40 is present on the same cell as the CDld. In some embodiments, the CDld / a- GalCer complex binds to a TCR while CD40 binds to CD40L present on the same cell as the TCR. In some aspects, when activated, NKT cells rapidly produce Thl and Th2 cytokines, including interferon-gamma (IFN-y) and interleukin-4 (IL-4). In some embodiments, the presentation of the a- GalCer itself is independent of MHC-I or MHC-II.

[0078] In some embodiments, the provided nanoparticles include a ligand for natural killer T (NKT) cells, such as a-GalCer. In some aspects, the a-GalCer of the provided nanoparticles can provide adjuvant activity in the subject administered the nanoparticle. Insome aspects, the nanoparticles provided herein include payloads such as nucleic acids encoding one or more vaccine epitopes (such as cancer neoantigen epitopes), or polypeptides comprising vaccine epitopes. In some aspects, the a-GalCer of the provided nanoparticles can provide adjuvant activity for the vaccination.

[0079] In some embodiments, a-GalCer is isolated from a natural source. In some embodiments, a-GalCer is synthetically produced to be incorporated into the nanoparticle. In some embodiments, as described herein, the nanoparticles comprising a-GalCer increases immune responses when administered to a subject, for example, mediating anti-tumor activity, vaccine immune response, or other therapeutic functions. In some embodiments, the a-GalCer mediates the anti-tumor activity or therapeutic function. In some embodiments, the therapeutic function is regulating an autoimmune disease.

[0080] In some embodiments, the a-GalCer promotes anti-tumor immunity. In some embodiments, the a-GalCer stimulates immune cells to kill cancer or tumor cells. In some embodiments, the a-GalCer stimulates and / or activates NKT cells, which then kill or destroy cancer or tumor cells. In some embodiments, the nanoparticle is delivered to the spleen. In some embodiments, after delivery to the spleen, the a-GalCer in the nanoparticle is presented to NKT cells through DCs. In some embodiments, after the a-GalCer is presented to NKT cells, the NKT cells mediate anti-tumor activity. In some embodiments, the nanoparticle is delivered to the tumor microenvironment (TME), and / or to tumor draining lymph nodes (TDLN). In some embodiments, after delivery to the TME or TDLN, the a-GalCer in the nanoparticle is presented to NKT cells through DC, to stimulate anti-tumor activity.B. Lipid Nanoparticles and Lipoplexes

[0081] Provided herein are nanoparticles, such as lipid nanoparticles (LNPs) comprising a- GalCer as a component. In some contexts, LNPs are nanoparticles comprising lipids which can act as pharmaceutical drug delivery systems, and in some cases as part of the pharmaceutical drug. In some aspects, LNPs are commonly used non-viral delivery’ system for therapeutics and vaccines. LNPs are typically spherical with the core of the LNP or a layer being lipophilic while the outside surface is often hydrophilic, allowing emulsions of the LNPs to be formed within an aqueous solution. In some contexts, the core can be lipophilic to allow for solubilization and delivery of lipophilic molecules as a payload. In some contexts, the LNP can include a bilayer, allowing the core to by hydrophilic, similar to the outside surface,allowing solubilization and deliver of hydrophilic molecules as a pay load. In some aspects, LNPs are often used with emulsifiers, which may include triglycerides, diglycerides, monoglycerides, fatty acids, steroids, and / or waxes, which can help to stabilize the LNPs while also preventing particle agglomeration. LNPs can be used to deliver a wide range of payloads (i.e., cargo) to cells, including drugs or nucleic acids.

[0082] In some embodiments, the provided nanoparticles are cationic nanoparticles. In some embodiments, the LNP is comprises lipids which are amphiphilic. In some embodiments, the lipids may include l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1,2- dioleoyl-sn- glycero-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-trimethylammonium- propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), 2.3-dioleyloxy-N-[2- (sperminecarboxamido)ethyl] -N,N-dimethyl- 1 -propanaminium trifluoroacetate (DOSP A), and / or 2- (((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)- 2,3,4,7,8,9,10,l l,12,13,14,15,16,17-tetradecahydro-lH-cyclopenta[a]phenanthren-3- y l)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methy lethan- 1 -aminium bromide (BHEM- Cholesterol). In some aspects, the provided nanoparticles comprise DOTMA and DOPE.

[0083] In some embodiments, the lipids may include analogues to any of the previously mentioned lipids. In some embodiments, the LNP includes a helper lipid, which may respond to pH changes during subcellular trafficking in endosomes. In some embodiments, the helper lipid may have an amino head group, phosphoethanolamine, and unsaturated oleoyl lipid chains. In some embodiments, the lipids may further include cholesterol. In some embodiments, DOTMA is a cationic lipid that helps in gene transfer. In some embodiments, is a quaternary' ammonium lipid. In some embodiments, DOTMA induces a positive charge on the surface which promotes efficient interactions between the nanoparticle and the cell surface.

[0084] In some embodiments, the provided nanoparticles comprise a nucleic acid as a payload. In some aspects, a nucleic acid is delivered via the provided nanoparticles. In some embodiments, the nanoparticle aids in delivery of the nucleic acid. In some embodiments, the nanoparticle leads to delivery of the nucleic acid to a cell. In some embodiments, the nanoparticle delivers the nucleic acid intracellularly. In some embodiments, the nanoparticle overcomes endosomal barriers for intracellular delivery of the nucleic acid. In some embodiments, the nanoparticle fuses with the cell membrane, leading to intracellular delivery of the nucleic acid.

[0085] In some embodiments the LNP is a lipoplex (LPX) nanoparticle. In some aspects, LPX are cationic lipid-nucleic acid complexes, formed by the interaction of anionic nucleic acids binding to positively charged lipid vesicles. In some embodiments, the LNP is an LPX nanoparticle comprising DOTMA and DOPE. In some aspects, the provided nanoparticles are LPX nanoparticles comprising DOTMA, DOPE and a-GalCer, and a nucleic acid payload, such as an RNA payload. In some aspects, the nucleic acid to be delivered using the nanoparticles provided herein include sequences encoding one or more vaccine epitopes.

[0086] In some embodiments, the provided nanoparticles can aid in treating cancer. In some embodiments, the cationic nanoparticles induce antigen specific CD4+ T cell proliferation thereby reducing cancer. In some embodiments, the CD4+ T cell proliferation is due to delivery of the nucleic acid to a target cell.

[0087] In some embodiments, the nanoparticle comprises a LNP. In some embodiments, nanoparticle comprises a LPX comprising a nucleic acid payload. In some embodiments, a nucleic acid is formulated in the nanoparticle. In some embodiments, a LPX nanoparticle formulation is used for delivery of nucleic acids. In some embodiments, a LPX nanoparticle formulation for the RNA (RNA-LPX) is used for deliver}' of any of the nucleic acids, such as RNA. In some aspects, the lipid nanoparticle is administered intravenously (IV). In some embodiments, a LPX nanoparticle formulation comprises cationic lipid l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and the phospholipid l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE). In some aspects, the DOTMA / DOPE liposomal component can be used for IV delivery and targeting of antigen-presenting cells.C. Method for Generation of Lipid Nanoparticles

[0088] In some aspects, provided are methods for generating the nanoparticles described herein. In some aspects, the nanoparticles are generated by mixing particular ratios of the different components of the provided nanoparticles.

[0089] In some embodiments, the nanoparticles are generated by mixing the 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and l,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) components at a particular ratio. In some embodiments, the ratio of DOTMA: DOPE is between about 5: 1 and about 1:5, such as about 3: 1 and about 1 :3; or about 2: 1 and about 1:2. In some embodiments, the ratio of DOTMA DOPE is between about3: 1 and about 1:3. In some embodiments, the ratio of DOTMA:DOPE is about 2: 1. In some embodiments, the ratio of DOTMA:DOPE is about 2: 1.

[0090] In some embodiments, the nanoparticles are generated by incorporating the a- GalCer into the DOTMA / DOPE nanoparticle at a particular proportion. In some embodiments, the proportion of a-GalCer is between about 0.1% and about 5% molar percentage, such as about 0.2%, 0.3%, 0.4%. 0.5%, 0.6%, 0.7%. 0.8%, 0.9%, 1.0%. 1.1%, 1.2%, 1.3%. 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.5%, 4.0%, 4.5%, or 5.0% molar percentage. In some embodiments, the proportion of a- GalCer is between about 0.5% and about 2% molar percentage. In some embodiments, the proportion of a-GalCer is about 0.5% molar percentage. In some embodiments, the proportion of a-GalCer is about 1 % molar percentage. In some embodiments, the proportion of a-GalCer is about 2% molar percentage.

[0091] In some aspects, the nanoparticle is a lipoplex (LPX). In some aspects, the LPX nanoparticle comprises a ribonucleic acid (RNA), for example, as a payload. In some aspects, the LPX nanoparticle is generated by incorporating the RNA at a particular charge ratio. In some embodiments, the nucleic acid is RNA, and the DOTMA:RNA charge ratio is between about 1 :2 to about 2:1, such as about 1.0:2, 1.1 :2, 1.2:2, 1.3:2, 1.4:2, 1.5:2, 1.6:2, 1.7:2, 1.8:2,I.9:2, 1.0: 1, 1.1: 1, 1.2: 1, 1.3: 1, 1.4: 1, 1.5: 1, 1.6: 1, 1.7: 1, 1.8: 1, 1.9: 1, or 2.0: 1. In some embodiments, the nucleic acid is RNA, and the DOTMA:RNA charge ratio is about 1.3:2.II. DELIVERY OF NUCLEIC ACIDS

[0092] In some embodiments, the provided nanoparticles comprise a nucleic acid. In some aspects, the nucleic acid is delivered to a subject as a payload. In some aspects, a nucleic acid is delivered via the provided nanoparticles. In some embodiments, the nanoparticle aids in delivery of the nucleic acid. In some aspects, the provided nanoparticle is a lipoplex (LPX) nanoparticle, such as a cationic lipid-nucleic acid complex.

[0093] As described herein, the provided nanoparticles comprising a-GalCer can be used to deliver various nucleic acid therapeutic modalities, including a ribonucleic acid (RNA), such as a circular RNA (circRNA), a messenger RNA (mRNA), a small inhibitory RNA (siRNA), an inhibitory RNA (RNAi), and / or a microRNA. In some aspects, the delivered nucleic acids, such as RNA, can enhance anti-tumor immune response or immune response against a vaccine epitope.

[0094] A. Nucleic Acids

[0095] In some aspects, delivery of nucleic acid to a target cell can be used for therapeutic potential. For instance, it can allow delivery of nucleic acids encoding a protein that stimulates the immune system near the target cell or leads the target cells to express the protein in such a way that immune cells kill the target cell. In some aspects, such delivery is beneficial in the context of treating or preventing tumors or cancer, for prophylactic uses or in the context of vaccination.

[0096] In some embodiments, the nanoparticle is used to deliver a nucleic acid. In some embodiments the nucleic acid is a ribonucleic acid (RNA). In some embodiments the RNA is a circRNA, an mRNA, a siRNA, an RNAi, and / or a microRNA. In some embodiments, the RNA enhances the anti-tumor effect or the therapeutic effect of the nanoparticle. In some embodiments, the nucleic acid is a deoxyribonucleic acid (DNA). In some embodiments, the RNA enhances the anti-tumor effect or the therapeutic effect of the nanoparticle.

[0097] siRNA or small interfering RNA or silencing RNA are RNA molecules that are double stranded and non-coding RNA molecules. They are typically 20-24 base pairs in length and are ty pically produced through the Dicer enzyme catalyzing production from long double stranded RNA molecules. siRNA can bind to mRNA with a complementary sequence and lead to mRNA degradation. Delivery of siRNA to a cell can be used to reduce specific mRNA transcripts, leading to decreased protein production from the mRNA. This is a form of RNA interference or RNAi. Another form of RNAi includes microRNA, which are similar to siRNAs but may contain post- transcriptional modifications. MicroRNAs also work to deplete target mRNAs and thereby decrease proteins produced by the mRNAs. Delivery of microRNAs to target cells offer therapeutic potentials by decreasing production of negative proteins.

[0098] mRNA is RNA that can be translated into a protein and its delivery into target cells can lead to localized production of protein within the target cell. mRNA is rapidly degraded and so needs to be efficiently delivered to the target cell. In some embodiments, the nanoparticle is used to deliver mRNA to a target cell.

[0099] In some embodiments, the nucleic acid is a linear RNA molecule. In some embodiments, the nucleic acid is a messenger RNA (mRNA) molecule. In some embodiments, the nucleic acid is a capped mRNA.

[0100] In some embodiments, the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2',3'-cyclic phosphate at the 3' terminus.

[0101] In some embodiments, the linear RNA is at least 600 nt, at least 1000 nt, or at least 1200 nt in length. In some embodiments, the linear RNA is less than 2000 nt in length. In some embodiments, the linear RNA is at least 600 nt but less than 2000 nt in length, at least 1000 nt but less than 2000 nt in length, at least 1200 nt but less than 2000 nt in length, at least 1400 nt but less than 2000 nt in length, at least 600 nt but less than 1400 nt in length, or at least 600 nt but less than 2000 nt in length.

[0102] CircRNA is a covalently closed continuous loop of single-stranded RNA. CircRNA can be divided into four categories including exonic circRNA (ecircRNA). circular intronic RNA (ciRNA), exon-intron circRNA (ElciRNA), and intergenic circRNA.

[0103] For delivery of RNA-encoded genes into cells and expression of the gene products, the stability of RNA is one of the critical factors in enhancing the gene product expression. Strategies have been developed to overcome the lack of stability and potential immunogenicity of linear mRNAs. Developed approaches include using untranslated regions (UTRs) such as those present in the native beta-globin mRNA, using methylguanosine cap analogs to protect the mRNA from de- capping enzymes, nucleoside modification, and codon optimization. However, only modest improvements have been observed.

[0104] The configuration of circRNA results in several advantages compared to linear mRNA, including resistance to exonuclease-mediated degradation, increased stability, extended half-life, increased protein expression, and reduced immunogenicity (Chen, RNA Biol, 12(4):381-388 (2015); Wesselhoeft et al., Nat Commun, 9(1):2629 (2018)). circRNA generally has a longer half- life compared to their linear mRNA counterparts (Wesselhoeft et al.. Nat Commun, 9(1):2629 (2018)). Accordingly, circRNA improves protein expression (e.g., of the encoded gene product) over its lifetime compared to linear mRNAs. CircRNA can be synthesized in vitro by chemical, enzymatic, and ribozymatic approaches. One approach, using permuted intron-exon (PIE) splicing to result in RNA circularization, has been developed to express gene products from circRNA. PIE splicing systems based on Group I introns that are naturally found in the rRNA, tRNA, and mRNA genes of bacteria and non-metazoan eukaryotes can produce circRNA by self-splicing.

[0105] In some embodiments, the circular RNA is at least 600 nt, at least 1000 nt, or at least 1200 nt in length. In some embodiments, the circular RNA is less than 2000 nt in length.In some embodiments, the circular RNA is at least 600 nt but less than 2000 nt in length, at least 1000 nt but less than 2000 nt in length, at least 1200 nt but less than 2000 nt in length, at least 1400 nt but less than 2000 nt in length, at least 600 nt but less than 1400 nt in length, or at least 600 nt but less than 2000 nt in length.

[0106] In some embodiments, the nucleic acid is a DNA molecule. In some embodiments, the nucleic acid is a naked DNA molecule. In some embodiments, the nucleic acid is a double-stranded DNA molecule. In some embodiments, the nucleic acid is a singlestranded DNA molecule. In some embodiments, the nucleic acid is a modified DNA molecule. In some embodiments, the nucleic acid is modified to enhance its stability. In some embodiments, the nucleic acid is a closed-ended DNA molecule. In some embodiments, the nucleic acid is a naked closed-ended DNA molecule.

[0107] In some embodiments, the DNA is at least 600 nt, at least 1000 nt, or at least 1200 nt in length. In some embodiments, the DNA is less than 2000 nt in length. In some embodiments, the DNA is at least 600 nt but less than 2000 nt in length, at least 1000 nt but less than 2000 nt in length, at least 1200 nt but less than 2000 nt in length, at least 1400 nt but less than 2000 nt in length, at least 600 nt but less than 1400 nt in length, or at least 600 nt but less than 2000 nt in length.B. Encoded Sequences

[0108] In some aspects, the nucleic acids delivered based on the provided nanoparticles and compositions include nucleic acid molecules that encode one or more exogenous molecules, such as for therapeutic or prophylactic uses. In some aspects, In some aspects, the nucleic acid comprises a coding sequence for a gene product (e.g., a protein) and the nucleic acid is delivered to a cell or a subject. In some aspects, provided nanoparticles are used to deliver the nucleic acid to a cell or a subject, and to express the exogenous molecules (e.g., gene product encoded by a coding sequence of the nuclei acid) in the cell or the subject. In some aspects, the nucleic acid also comprises gene coding sequences and / or other elements that may be required for transcription, translation, and / or expression of the encoded gene product.

[0109] In some embodiments, the nucleic acid is at least about 50 nucleotides (nt), 750 nt, 1000 nt, 1250 nt, 1500 nt, 2000 nt, 2500 nt, 3000 nt, 3500 nt, 4000 nt, 4500 nt, 5000 nt, 6000 nt, 7000 nt, 8000 nt, 9000 nt, or 10000 nt in length. In some embodiments, the nucleic acid is at least about 2000 nt in length. In some embodiments, the nucleic acid is at least about3000 nt in length. In some embodiments, the nucleic acid is at least about 4000 nt in length. In some embodiments, the nucleic acid is at least about 5000 nt in length.

[0110] In some aspects, the nucleic acid used with the provided nanoparticles, comprise a sequence encoding one or more exogenous molecules. In some aspects, the one or more exogenous molecules (e.g., gene product encoded by a coding sequence) includes one or more of: a vaccine antigen, a cancer antigen, a CRISPR system, including a nuclease and / or a guide RNA (gRNA), a nuclease, a therapeutic polypeptide, an antibody or antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.[OHl] In some embodiments, the exogenous molecule comprises a vaccine epitope. In some embodiments, the nucleic acid encodes a vaccine epitope. In some embodiments, the nucleic acid encodes one or more vaccine epitopes. In some embodiments, the vaccine epitope comprises an infectious disease vaccine epitope. In some embodiments, the vaccine epitopes are bacterial epitopes, viral epitopes, cancer epitopes, fungal epitopes, and / or infectious disease epitopes.

[0112] In some embodiments, the vaccine epitope comprises a viral vaccine epitope. In some aspects, the viral vaccine epitope comprises an epitope encoded by a virus. In some aspects, the viral vaccine epitope is from a virus that is a member of Coronaviridae. In some embodiments, the viral vaccine epitope is from an alphacoronavirus, a betacoronavirus, a deltacoronavirus, or a gammacoronavirus. In some embodiments, the virus is a human coronavirus OC43 (HCoV-OC43), human coronavirus HKU1 (HCoV-HKUl), human coronavirus 229E (HCoV-229E), human coronavirus NL63 (HCoV-NL63), Middle East respiratory syndrome-related coronavirus (MERS- CoV), severe acute respiratory' syndrome coronavirus (SARS-CoV), or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In some embodiments, the virus is SARS-CoV-2. In some aspects, the viral vaccine epitope is from a SARS-CoV-2 vims.

[0113] In some embodiments, the vaccine epitopes are infectious disease epitopes. In some embodiments, the epitope is a disease-associated epitope, i.e.. an epitope that arises from presentation of a viral or bacterial antigen, or part of a bacterial or viruses such as a protein or peptide expressed in a bacterial or viruses which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens of bacterial or viruses. For example, infectious disease epitopes include the SARS- CoV-2 spike glycoprotein.

[0114] In some embodiments, the vaccine epitope comprises a cancer epitope. In some aspects, the cancer epitope is a tumor-associated antigens (TAAs) or a tumor-specific antigens (TSAs). Exemplary cancer antigens, TAAs or TSAs include, but are not limited to, those described in, for example, Liu et al., Journal of Hematology & Oncology 15:28 (2022); Buonaguro et al.. Vaccines (Basel). 2020 Dec; 8(4): 615; and Zhao et al., Vaccines (Basel). 2021 Feb; 9(2): 85.

[0115] In some embodiments, the vaccine epitope comprises a cancer epitope. In some embodiments, the cancer epitope is a cancer neoantigen epitope.

[0116] In some embodiments, the step of identifying infectious disease-associated antigens and epitopes or identifying sequence differences involves using next generation sequencing (NGS). In some embodiments, the step of identifying infectious disease-associated epitopes or identifying sequence differences comprises sequencing genomic DNA and / or RNA of the virus or bacteria sample.

[0117] In some embodiments, the vaccine antigen comprises a cancer antigen. In some aspects, the cancer antigen is a tumor-associated antigens (TAAs) or a tumor-specific antigens (TSAs). Exemplary cancer antigens, TAAs or TSAs include, but are not limited to, those described in, for example, Liu et al., Journal of Hematology & Oncology 15:28 (2022); Buonaguro et al.. Vaccines (Basel). 2020 Dec; 8(4): 615; and Zhao et al., Vaccines (Basel). 2021 Feb; 9(2): 85.

[0118] In some embodiments, the vaccine epitopes are cancer epitopes. In some embodiments, the epitope is a tumor-associated epitope, i.e., an epitope that arises from presentation of a tumor antigen, or part of a tumor cell such as a protein or peptide expressed in a tumor cell which may be derived from the cytoplasm, the cell surface or the cell nucleus, in particular those which primarily occur intracellularly or as surface antigens of tumor cells. In some embodiments, a tumor-associated epitope preferably comprises any epitope which is expressed in and optionally characteristic with respect to type and / or expression level for tumors or cancers as well as for tumor or cancer cells.

[0119] In some embodiments, the cancer specific mutations include mutations present in one or more cancer cells of a patient. In some aspects, the vaccine epitope is a cancer neoantigen epitope (also called neoepitope or cancer neoepitope). In some embodiments, the cancer neoepitope is a new epitope, e.g.. a newly arising peptide sequence, that arises in cancer cells as a result of mutations in the cancer cell. In some aspects, cancer neoepitopes of theprovided embodiments can be selected by identifying of a number of mutations which provides a sufficient number of neoepitopes to be included encoded by the nucleic acid payload. A “cancer mutation” relates to a sequence difference between the nucleic acid contained in a cancer cell and the nucleic acid contained in a normal cell.

[0120] In some cases, the cancer mutations are non-synonymous mutations, in some cases non- synonymous mutations of proteins expressed in a tumor or cancer cell. In some embodiments, cancer specific somatic mutations or sequence differences are determined in the genome, in some cases the entire genome, of a tumor specimen. In some aspects, neoepitopes can be identified from the cancer mutation signature of the genome, in some cases the entire genome of one or more cancer cells. In some embodiments, identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identifying the genome-wide cancer mutation profile. In some embodiments, cancer specific somatic mutations or sequence differences are determined in the exome, in some cases the entire exome, of a tumor specimen. In some aspects, neoepitopes can be identified from the cancer mutation signature of the exome, in some cases the entire exome of one or more cancer cells. In some embodiments, identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identify ing the exome-wide cancer mutation profile. In some embodiments, cancer specific somatic mutations or sequence differences are determined in the trans criptome, in some cases the entire transcriptome, of a tumor specimen. In some aspects, neoepitopes can be identified from the cancer mutation signature of the trans criptome, in some cases the entire transcriptome of one or more cancer cells. In some embodiments, identifying cancer specific somatic mutations in a tumor specimen of a cancer patient comprises identify ing the transcriptome-wide cancer mutation profile. In some embodiments, identifying cancer specific somatic mutations or identify ing sequence differences comprises single cell sequencing of one or more, in some cases 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or even more cancer cells. In some aspects, neoepitopes can be identified from a cancer mutation signature of said one or more cancer cells.

[0121] In some embodiments, the cancer cells from which cancer neoepitopes can be identified include circulating tumor cells. The cancer cells such as the circulating tumor cells may be isolated prior to single cell sequencing. In some embodiments, identifying cancer specific somatic mutations or identifying sequence differences involves using next generation sequencing (NGS). In some embodiments, identifying cancer specific somatic mutations oridentifying sequence differences comprises sequencing genomic DNA and / or RNA of the tumor specimen.

[0122] To reveal cancer specific somatic mutations or sequence differences, and to determine cancer neoepitopes to be encoded by the nucleic acid payload, the sequence information obtained from the tumor specimen is compared with a reference such as sequence information obtained from sequencing nucleic acid such as DNA or RNA of normal non- cancerous cells such as germline cells which may either be obtained from the patient or a different individual. In some embodiments, normal genomic germline DNA is obtained from peripheral blood mononuclear cells (PBMCs)

[0123] In some aspects, the nucleic acid encodes neoepitopes (i.e., tumor-associated antigens (TAAs) that are specifically expressed in the patient’s cancer) identified as having potential immunostimulatory activities.

[0124] In some embodiments, the nucleic acid encodes a cancer neoantigen epitopes selected from among one or more of MEI, M26, Gludl, Mtchl, M18, Steap2, and E2f8.

[0125] In some embodiments, the vaccine epitope is an epitope of an autoantigen or an antigen associated with an autoimmune disease. Autoimmune diseases are broadly classified into two categories, organ-specific and systemic diseases. The precise etiology of systemic auto-immune diseases is not identified. In contrast, organ-specific auto-immune diseases are related to a specific immune response including B and T cells, which targets the organ and thereby induces and maintains a chronic state of local inflammation. Examples of organspecific auto-immune diseases include type 1 diabetes, myasthenia gravis, thyroiditis and multiple sclerosis. In some aspects, in these conditions, a single or a small number of autoantigens have been identified, including insulin, the acetylcholine muscle receptor, thyroid peroxidase and major basic protein, respectively. In some embodiments, the vaccine epitope includes an epitope from insulin, the acetylcholine muscle receptor, thyroid peroxidase and / or major basic protein. Autoimmune diseases or disorders include diseases that result from an aberrant immune response of an organism against its own cells and tissues due to a failure of the organism to recognize its own constituent parts (down to the sub- molecular level) as "‘self’.

[0126] In some aspects, the nucleic acid encodes multiple vaccine epitopes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 28, 29, or 30 epitopes. In some aspects, the nucleic acid encodes 2 different vaccine epitopes. In some aspects, the nucleic acid encodes 3 different vaccine epitopes. In some aspects, the nucleic acidencodes 4 different vaccine epitopes. In some aspects, the nucleic acid encodes 5 different vaccine epitopes. In some aspects, the nucleic acid encodes 6 different vaccine epitopes. In some aspects, the nucleic acid encodes 7 different vaccine epitopes. In some aspects, the nucleic acid encodes 8 different vaccine epitopes. In some aspects, the nucleic acid encodes 9 different vaccine epitopes.

[0127] In some aspects, when the nucleic acid encodes multiple epitopes, each of the different vaccine epitopes are from different proteins. In some aspects, when the nucleic acid encodes multiple epitopes, each of the different vaccine epitopes are from the same protein. In some aspects, when the nucleic acid encodes multiple epitopes, some of the vaccine epitopes can be from the same protein, and others can be from different proteins.

[0128] In some embodiments, the exogenous molecule comprises one or more components of a CRISPR system. In some aspects, the term “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated C‘Cas”) genes, including sequences encoding a Cas gene, and various nucleic acid sequences associated with a Cas nuclease, such as a guide RNA (gRNA), and / or other sequences and transcripts from a CRISPR locus. In some embodiments, the CRISPR system includes a sequence-specific nuclease. In some embodiments, the sequencespecific nuclease is a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Casl2 nuclease or a Casl3 nuclease. In some aspects, the CRISPR system includes a non-coding guide RNA (gRNA), which sequence- specifically binds to DNA, and a Cas nuclease (e.g, Cas9, CasX, Casl2, or Casl3), with a sequence-specific nuclease functionality. In some aspects, a gRNA is a nucleic acid that promotes the specific targeting or homing of a gRNA molecule / Cas nuclease complex to a target nucleic acid, such as a locus on the genomic DNA of a cell. In some embodiments, the exogenous molecule comprises a guide RNA (gRNA). In some aspects, the exogenous molecule encoded by sequences in the insert sequences include a Cas nuclease. In some embodiments, the Cas nuclease is a Cas9 nuclease. In some embodiments, the Cas nuclease is a CasX nuclease. In some embodiments, the Cas nuclease is a Cas 12 nuclease. In some embodiments, the Cas nuclease is a Cas 13 nuclease. Exemplary Cas nucleases that can be encoded in the provided RNA molecules, including circRNA molecules, include, but are not limited to, those described in, for example, Jinek et al., Science, 343(6176): 1247997, 2014; Nishimasu et al., Cell, 156:935-949, 2014; Cong et al., Science 2013, 399(6121):819-823; Wang et al., Cell 2013, 153(4):910-9I8; Mali et al., Science2013. 399(6121):823-826; Cebrian-Serrano et al., Mamm Genome. 2017; 28(7): 247-261; Collias et al., Nature Communications 12:555 (2021); and Chen et al.. Innovation (Camb). 2022 Jul 12; 3(4): 100264

[0129] In some embodiments, the exogenous molecule comprises an antibody or an antigen- binding fragment thereof. In some aspects, exemplary antibody or antigen-binding fragment thereof that can be encoded in the provided RNA molecules, including circRNA molecules, include any known therapeutic antibodies, for example, those described in The Therapeutic Structural Antibody Database (Thera-SAbDab); Raybould et al., Nucleic Acids Research, 2020, 48(D1): D383-D388,; http: / / opig.stats.ox.ac.uk / webapps / therasabdab.

[0130] In some embodiments, the exogenous molecule comprises an exogenous molecule comprises an immunomodulatory polypeptide. In some aspects, the immunomodulatory polypeptide is selected from among an adjuvant, an immune checkpoint inhibitor, a cytokine or any combination thereof. In some embodiments, the immunomodulatory polypeptide comprises a cytokine. In some embodiments, the immunomodulatory’ polypeptide is selected from among IL-1, IL-la, IL-2. IL-3. IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10. IL-11, IL-12, IL-15, interferon (IFN)-a, IFN- , IFN-y, tumor necrosis factor (TNF)-a, TNF-(3, proteins in the TNF superfamily, human growth hormone, N-methionyl human growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones such as follicle stimulating hormone (FSH). thyroid stimulating hormone (TSH). and luteinizing hormone (LH), hepatic growth factor, fibroblast growth factor (FGF), prolactin, placental lactogen, tumor necrosis factor-a and -[k mullerian-inhibiting substance, mouse gonadotropin-associated peptide, inhibin, activin, vascular endothelial growth factor (VEGF), integrin, thrombopoietin (TPO), nerve growth factors (NGF)-[3. platelet-growth factor, transforming growth factor (TGF)-a, TGF-0, insulin-like growth factor (IGF)-l, IGF-2, erythropoietin (EPO), osteoinductive factors, macrophage-CSF (M-CSF), granulocytemacrophage- CSF (GM-CSF), granulocyte-CSF (G-CSF), leukemia inhibitory’ factor (LIF), kit ligand (KL) and / or a portion and / or combination thereof.

[0131] In some embodiments, the exogenous molecule comprises a transcription factor. In some aspects, exemplary transcription factors that can be encoded in the provided RNA molecules, including circRNA molecules, include those described in, for example, Becskei et al., Molecules. 2020 Apr; 25(8): 1902 or Pandelakis et al., Cell Systems (2020) 10(1): 1-14; and / or zinc finger protein (ZFP) or transcription activator-like effectors (TALEs) proteins thatcan be engineered to target specific sequences, for example, as described in US 6,140,081; US 6,453,242; US 6,534,261; WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536; WO 03 / 016496; US 20110301073; and Gaj etal., Trends in Biotechnology, 2013, 31(7), 397-405.

[0132] In some embodiments, the exogenous molecule comprises a reporter molecule. In some embodiments, the reporter molecule is a detectable protein, such as a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (eGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP). and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, codon- optimized, stabilized and / or enhanced variants of the fluorescent proteins. In some aspects, the reporter molecule is an enzyme, such as a luciferase, including a firefly luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyl transferase (CAT). Exemplary’ light-emitting reporter molecules include luciferase (luc), firefly luciferase P-galactosidase. chloramphenicol acetyltransferase (CAT). - glucuronidase (GUS) or variants thereof. In some aspects, expression of the enzyme can be detected by addition of a substrate that can be detected upon the expression and functional activity of the enzyme. In some embodiments, the reporter molecule comprises an eGFP. In some embodiments, the reporter molecule comprises an RFP.

[0133] In some embodiments, the nucleic acid further comprises one or more elements that facilitates the expression and / or processing of the encoded gene products, such as exogenous molecules.

[0134] In some embodiments, the nucleic acid further comprises a translation initiation element. In some embodiments, the translation initiation element is an internal ribosome entry site (IRES) or a Translation Initiator of Short 5' UTR (TISU) element. In some embodiments, the translation initiation element is or comprises an internal ribosome entry site (IRES). In some embodiments, the IRES comprises a Coxsackievirus B3 internal ribosome entry site (CVB3 IRES) sequence.

[0135] In some embodiments, the translation initiation element comprises a Translation Initiator of Short 5' UTR (TISU) element. TISU is a regulatory element that controls both transcription and translation initiation of genes with basic cellular functions.

[0136] In some cases, when the insert sequence contains sequences encoding multiple exogenous molecules, a multicistronic element can be used, typically placed between the different coding sequences, to express multiple polypeptide gene products. Exemplary multicistronic elements include 2A elements that result in the separation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe, Genetic Vaccines and Ther. 2: 13 (2004) and de Felipe et al. Traffic 5:616-626 (2004)).

[0137] In some embodiments, the nucleic acid comprises a poly adenine cytosine (poly AC) sequence. In some aspects, the poly AC sequence can be located 3' of the coding sequence for the exogenous molecule.III. THERAPEUTIC AND PROPHYLACTIC METHODS AND USES

[0138] Also provided are methods of administering and uses, such as prophylactic and therapeutic uses, of the provided nanoparticles, or compositions comprising the nanoparticle. In some embodiments, nanoparticle and / or composition is administered in an effective amount to effect treatment of the disease or disorder. Uses include uses of nanoparticle and / or composition in such methods and treatments, and in the preparation of a medicament in order to cany7out such therapeutic or prophylactic methods. In some embodiments, the methods are carried out by administering nanoparticle and / or composition, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject. In some embodiments, the disease condition or disorder in the subject is cancer. In some embodiments, the disease condition or disorder in the subject is an infectious disease, optionally a viral infection. In some embodiments, the disease condition or disorder in the subject is an autoimmune disease.

[0139] In some aspects, the a-GalCer component of the nanoparticle stimulates an immune response, and also allows selective targeting of the payload contained in the nanoparticle to particular regions or areas of a subject, after administration of the drug. In some aspects, also provided are methods and uses, including prophylactic and therapeutic methods and uses, such as vaccination methods, that employ the provided nanoparticles.

[0140] In some aspects, the provided nanoparticles can be used to treat or ameliorate diseases or conditions such as a cancer or a tumor, for example, by specifically delivering a payload, such as a nucleic acid drug, to the target site, such as a tumor or a tumor-draining lymph node, and in addition, by the action of the nucleic acid payload. For example, variousnucleic acid based drugs, such as small inhibitory RNAs (siRNAs) and microRNAs that block the expression of immunosuppressive proteins, as well as circular RNAs or mRNAs that encode immunostimulatory proteins such as cytokines and molecules of the tumor necrosis factor (TNF) superfamily.

[0141] In some embodiments, administration of nanoparticle or composition induces an immune response. In some embodiments, administration of nanoparticle or composition induces an immune response comprising a CD4+ T cell response, CD8+ T cell response and / or antibody response. In some embodiments, administration of nanoparticle or composition induces an immune response comprising a CD4+ T cell response and / or CD8+ T cell response. In some aspects, provided are nanoparticles that can enhance the efficiency of vaccine epitope presentation. In some aspects, the provided nanoparticles comprise a nucleic acid encoding one or more vaccine epitopes.

[0142] In some embodiments, the provided nanoparticles results in induction of an immune response such as a CD4+ T cell response, CD8+ T cell response and / or antibody response, specific for the vaccine epitopes.

[0143] In some aspects, the provided nanoparticles comprise a nucleic acid encoding one or more various vaccine epitopes, including cancer neoantigen epitopes, tumor-associated antigen (TAA) epitopes, and viral oncogene epitopes, for stimulating epitope-specific T cells. In some aspects, the provided embodiments can be used to control autoimmune diseases by promoting autoantigens-specific regulatory T cells to induce immune tolerance.

[0144] In some aspects, vaccine approaches that primarily target T cells and can elicit a robust T cell response are more efficient in controlling viruses that have more frequent mutants than antibody-specific vaccines.

[0145] In some aspects, the provided embodiments can be used to treat, prevent, reduce the severity of and / or vaccinate against a disease or disorder.

[0146] In some embodiments, the disease or disorder is an infectious disease. In some embodiments, the disease or disorder is a viral infection.

[0147] In some embodiments, the disease or disorder is a cancer or a tumor. Among the diseases or conditions that can be treated include, but are not limited to, B cell leukemia, lymphoma, B cell chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML), acute lymphocytic leukemia (ALL), Burkit's Lymphoma, mantle cell lymphoma (MCL), non-small cell lung cancer (NSCLC). neuroblastoma, renal cell carcinoma, colon cancer, colorectalcancer. breast cancer, epithelial squamous cell cancer, melanoma, myeloma, stomach cancer, brain cancer, lung cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, prostate cancer, testicular cancer, thyroid cancer, uterine cancer, adrenal cancer and head and neck cancer. In some aspects, the cancer is melanoma, non-small cell lung cancer, bladder cancer, colorectal cancer, triple negative breast cancer, renal cancer, and head and neck cancer. In some embodiments, the cancer is locally advanced or metastatic melanoma, non- small cell lung cancer, bladder cancer, colorectal cancer, triple negative breast cancer, renal cancer, or head and neck cancer.

[0148] In some embodiments, the cancer is colorectal carcinoma. In some embodiments, the cancer is carcinoma. In some embodiments, the carcinoma is metastatic or unresectable locally advanced melanoma. In some embodiments, the carcinoma is stage IV carcinoma. In some embodiments, the carcinoma is stage IIIC or stage IIID carcinoma. In some embodiments, the carcinoma is unresectable or metastatic carcinoma.

[0149] In some aspects, a-GalCer can also regulate autoimmune responses, the provided nanoparticles can be used to treat, prevent, delay the development of or reduce the severity of autoimmune diseases, for example by inducing immune tolerance by delivering various RNA therapeutics. In some embodiments, the disease or disorder is an autoimmune disease. In some embodiments, the autoimmune disease or disorder includes type 1 diabetes, my asthenia gravis, thyroiditis and multiple sclerosis.A. Localization of Lipid Nanoparticles

[0150] In some embodiments, the nanoparticles and compositions provided herein, when administered to a subject, can be targeted to or localized to a particular part of the subject’s body. In some embodiments, the nanoparticle is localized to the spleen. In some embodiments, the nanoparticle is localized to a tumor. In some embodiments, the nanoparticle is localized to a tumor- draining lymph node (TDLN).

[0151] In some contexts, lymph nodes are organs of the lymphatic system and a component of the adaptive immune system. In some cases, lymph nodes comprise an outer cortex and an inner medulla. Lymph nodes house lymphocytes, including B and T cells, and are linked throughout the body by lymphatic vessels. Lymph nodes act as filters for foreign particles, where particles are presented to lymphocytes, which may activate the lymphocytes to help coordinate an immune response to the foreign particles. In some aspects, the activation ofcertain lymphocytes, like B cells and T cells, is also helped within lymph nodes due to the dendritic cells and other antigen presenting cells present. Antigens from throughout the body may be caried from their location of origin to a lymph node by lymphatic flow draining into the lymph node through lymphatic vessels, allowing for the activation of immune cells present within the lymph node.

[0152] In some cases, lymph nodes that have lymphatic flow draining into them from a tumor are called tumor draining lymph nodes. Tumor draining lymph nodes can be essential in an effective anti-tumor T cell immune response, but immune suppressive factors may render the tumor draining lymph nodes to be compromised, leading to tumors evading the immune system or even metastasizing through the lymphatic system. Immunosuppressive factors can include extracellular vesicles, IL-6, TGF-0, prostaglandin-E2, and VEGF, which can suppress dendritic cells and antigen presentation, limiting the lymphocyte activation to cancer or tumors. In some cases, tumor draining lymph nodes may go through alterations, including lymphangiogenesis, blood vessel remodeling, and increased cytokine secretion, which may lead to a more tumor- supportive environment allowing for metastasis of the tumor to the tumor draining lymph node.

[0153] In some contexts, tumor draining lymph nodes can act as a promising site for immunotherapy to address cancer or tumors. Lymphocytes, such as cytotoxic T cells, within the tumor draining lymph node can be primed against cancer with the introduction of an antigen or epitope (e.g.. cancer neoantigen epitope) to the cells through presentation from antigen presenting cells. The introduction of an antigen or epitope (e g., cancer neoantigen epitope) and the priming of lymphocytes can lead the lymphocytes to become activated against cancer or tumor cells, thereby killing the cells, reducing the cancer or tumor burden.

[0154] In some embodiments, the provided nanoparticle targets the tumor draining lymph node, in some cases leading to accumulation of the provided nanoparticle or delivery of the provided nanoparticle to cells within the tumor draining lymph node. In some embodiments, the provided nanoparticle contains a nucleic acid that encodes an antigen or a vaccine epitope (e.g., cancer neoantigen epitope). In some embodiments, the provided nanoparticle targets antigen presenting cells and / or dendritic cells, leading the cells to present the antigen or the neoantigen. In some embodiments, the antigen or vaccine epitope (e g., cancer neoantigen epitope) encoded by a nucleic acid in the provided nanoparticle activates lymphocytes within the tumor draining lymph node. In some embodiments, the provided nanoparticle with a nucleic acid encoding an antigen or vaccine epitope (e.g., cancer neoantigen epitope) results in adecreased tumor burden compared the same nucleic acid being delivered using a different nanoparticle, or subjects that did not receive the nanoparticle.B. Compositions and Administration

[0155] Provided herein are compositions comprising the nanoparticles, such as nanoparticles including a nucleic acid. In some aspects, the nanoparticle is administered to a subject, such as a subject with a disease or condition, or to prevent or reduce the severity of a disease or condition. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition comprises a pharmaceutically acceptable excipient.

[0156] In some embodiments the LNP is a lipoplex (LPX) nanoparticle. In some aspects, LPX are cationic lipid-nucleic acid complexes, are formed by the interaction of anionic nucleic acids binding to positively charged lipid vesicles. In some embodiments, the LNP is an LPX nanoparticle comprising DOTMA and DOPE. In some aspects, the nanoparticles are LPX nanoparticles comprising DOTMA, DOPE, and a nucleic acid, such as an RNA encoding an exogenous molecule. In some aspects, the nucleic acid to be delivered using the nanoparticles include sequences encoding one or more vaccine epitopes.

[0157] In some embodiments, the nanoparticles, or compositions comprising the same, can be administered by any suitable means, for example, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectival injection, subconjuntival injection, sub-Tenon's injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary. and intranasal, and, if desired for local treatment, intralesional administration. In some embodiments, the nanoparticle is administered (e.g., in a lipoplex particle or liposome) intravenously, intramuscularly, subcutaneously, topically, orally , trans dermally, intraperitoneally , intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, intracranial, intrathoracic, or subcutaneous administration. Dosing and administration may depend in part on whether the administration is brief or chronic. Various dosing schedules include but are not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion.In some embodiments, the nanoparticles, or compositions comprising the same are administered intravenously.

[0158] In some embodiments, the nanoparticles or compositions is administered in the presence of an adjuvant. In some embodiments, the nanoparticles or compositions can be administered by repeat administration of the peptide a plurality of times. In some embodiments, repeated administration is with a low dose of peptide. In some embodiments, the repeat administration of the nanoparticles or compositions occurs over an extended time period, such as for up to three days, four days, five days, six days, one week, two weeks, three weeks or a month. In some embodiments, the nanoparticles or compositions can be administered in a high dose amount. In some embodiments, one or more administration modes, schedules or frequency of administration are employed.

[0159] In some aspects, the nanoparticles in the composition are engineered to promote expression of epitopes in the antigen presenting cell. Other methods that can be used to deliver nucleic acids to the subject include viral, e.g., retroviral or lentiviral, transduction, transposons, and electroporation.

[0160] In some embodiments, nucleic acids are transferred into cells using recombinant infectious virus particles, such as, e.g., vectors derived from simian virus 40 (SV40), adenoviruses, adeno-associated virus (AAV). In some embodiments, nucleic acids are transferred into T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors.

[0161] Pharmaceutically acceptable excipients can also include solvents, bulking agents, buffering agents, tonicity adjusting agents, and preservatives. In some embodiments, the compositions comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).C. Vaccination Methods and Prophylactic Methods

[0162] Provided herein are methods of administering and uses, such as in prophylactic methods and / or vaccination methods, of the provided nanoparticles and / or compositions. Also provided are methods of vaccinating a subject that involves administering the provided nanoparticles and / or compositions provided herein. Also provided are methods of enhancing vaccine epitope presentation to elicit an immune response that involves administering theprovided nanoparticles and / or compositions. In some aspects any of the provided methods and uses, the provided nanoparticles and / or compositions are administered to a subject for vaccination and / or to prevent, delay the development of or progression of, or reduce the severity of a disease or condition.

[0163] In some embodiments, the nanoparticles and / or compositions induce an increased immune response to the vaccine epitope compared to a subject that without the administration of the nanoparticles or compositions. In some embodiments, the nanoparticles comprising nucleic acids encoding exogenous molecules, such as one or more vaccine epitopes or compositions induce an immune response comprising a CD4+ T cell response, CD8+ T cell response and / or antibody response. In some embodiments, the nanoparticles and / or compositions prevents or reduces the severity of a disease or disorder associated with the vaccine epitope in the subject.

[0164] In some embodiments, the nanoparticles and / or compositions is administered to a subject for vaccinating the subject against a disease or disorder. In some aspects, one or more vaccines may be administered to a subject, e.g.. subject is administered one vaccine with a combination of vaccine epitopes and also administered a separate vaccine with a different combination of vaccine epitopes

[0165] In some embodiments, the vaccine is administered such that it is delivered to the spleen. For example, the vaccine can be administered such that one or more epitopes (e.g.. neoepitopes) are delivered to antigen presenting cells, for example, in the spleen.

[0166] In some embodiments, an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle. In some embodiments, the immune response comprises CD4+ T cell response, CD8+ T cell response and / or antibody response. In some embodiments, the immune response comprises CD4+ T cell response or CD8+ T cell response.D. Therapeutic Methods

[0167] Also provided are methods of administering and uses, such as therapeutic uses, of the nanoparticles, such as those comprising nucleic acids encoding exogenous molecules, such as one or more vaccine epitopes, or compositions comprising the same.

[0168] In some embodiments, the provided nanoparticles and / or compositions are employed in a method of treatment for a subject having a disease or disorder. In some aspects.the methods and uses can be employed to treat, reduce the symptoms of, or ameliorate the disease or condition.

[0169] In some embodiments of the methods, a nanoparticles or compositions is administered in a way that enhances the potential for an immune response, such as an immune response against the disease or disorder or particular target cells involved in the disease or disorder. In some aspects, this enhancement is by the induction of epitope-specific priming to particular target proteins or cells.E. Combination Therapy

[0170] Also provided are methods of administering and uses, such as in combination therapy, of the nanoparticles encoding the exogenous molecule, or compositions comprising the same.

[0171] In some embodiments, the nanoparticles, such as those comprising nucleic acids encoding exogenous molecules, such as one or more vaccine epitopes, or compositions comprising the same is administered in an effective amount in combination with an additional therapy to effect treatment of the disease or disorder. Such methods and uses include therapeutic methods and use, for example, involving administration of the nanoparticles, or compositions, to a subject having a disease, condition, or disorder expressing or associated with cancer or autoimmune disease. In some embodiments, the methods are carried out by administering the nanoparticles comprising nucleic acids encoding the exogenous molecule, or compositions in combination with an additional therapy, to the subject having, having had, or suspected of having the disease or condition. In some embodiments, the methods thereby treat the disease or condition or disorder in the subject.

[0172] In some embodiments, the additional agent for combination treatment or combination therapy enhances, boosts and / or promotes the efficacy and / or safety of the therapeutic effect of the nanoparticles, or compositions. In some embodiments, the additional agent can treat the same disease, condition or a comorbidity.

[0173] In some embodiments, the additional therapeutic agent is an immune checkpoint inhibitor. In some embodiments, the additional therapeutic agent is PD-1 axis binding antagonist.

[0174] In some aspects, a checkpoint inhibitor targets at least one immune checkpoint protein to alter the regulation of an immune response. Immune checkpoint proteins include,e.g., CTLA-4, PD-L1, PD-1. PD-L2, VISTA, B7-H2, B7-H3. B7-H4. B7-H6, 2B4. ICOS, HVEM, CEACAM, LAIR1, CD80, CD86, CD276, VTCN1, MHC class I, MHC class II, GALS, adenosine, TGFR, CSF1R, MICA / B, arginase, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPalpha (CD47), CD48, 2B4 (CD244), B7.1. B7.2, ILT-2, ILT-4, TIGIT, LAG-3. BTLA. IDO, 0X40, and A2aR. In some embodiments, molecules involved in regulating immune checkpoints include, but are not limited to: PD-1 (CD279), PD-L1 (B7-H1, CD274), PD-L2 (B7- CD, CD273), CTLA-4 (CD 152), HVEM, BTLA (CD272), a killer-cell immunoglobulin-like receptor (KIR), LAG-3 (CD223), TIM-3 (HAVCR2), CEACAM, CEACAM- 1, CEACAM-3, CEACAM-5, GAL9, VISTA (PD-1H), TIGIT, LAIR1, CD160, 2B4, TGFRbeta, A2AR, GITR (CD357), CD80 (B7- 1), CD86 (B7-2), CD276 (B7-H3), VTCNI (B7-H4), MHC class I, MHC class II, GALS, adenosine, TGFR, B7-H1, 0X40 (CD134), CD94 (KLRD1), CD137 (4-1BB), CD137L (4- 1BBL). CD40, IDO, CSF1R, CD40L, CD47, CD70 (CD27L), CD226, HHLA2, ICOS (CD278), ICOSL (CD275), LIGHT (TNFSF14, CD258), NKG2a, NKG2d. OX40L (CD134L), PVR (NECL5. CD 155), SIRPa, MICA / B. and / or arginase. In some embodiments, an immune checkpoint inhibitor (i.e., a checkpoint inhibitor) decreases the activity of a checkpoint protein that negatively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In other embodiments, a checkpoint inhibitor increases the activity of a checkpoint protein that positively regulates immune cell function, e.g., in order to enhance T cell activation and / or an anti-cancer immune response. In some embodiments, the checkpoint inhibitor is an antibody. Examples of checkpoint inhibitors include, without limitation, a PD-1 axis binding antagonist, a PD-L1 axis binding antagonist (e.g., an anti-PD-Ll antibody, e.g., atezolizumab (MPDL3280A)), an antagonist directed against a co-inhibitory molecule (e.g., a CTLA-4 antagonist (e.g., an anti- CTLA-4 antibody), a TIM-3 antagonist (e.g., an anti-TIM-3 antibody), or a LAG-3 antagonist (e.g., an anti-LAG-3 antibody)), or any combination thereof. In some embodiments, the immune checkpoint inhibitors comprise drugs such as small molecules, recombinant forms of ligand or receptors, or antibodies, such as human antibodies (see. e.g., WO 2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012). In some embodiments, know n inhibitors of immune checkpoint proteins or analogs thereof may be used, in particular chimerized, humanized or human forms of antibodies may be used.

[0175] For example, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PDL1 binding antagonist and a PDL2 binding antagonist. Alternative names for “PD-1”include CD279 and SLEB2. Alternative names for “PD-L1” include B7-H1, B7-4, CD274, and B7-H. Alternative names for “PD-L2” include B7-DC, Btdc, and CD273. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

[0176] Exemplary7immune checkpoint inhibitors include Tremelimumab (CTLA-4 blocking antibody, also known as ticilimumab, CP-675.206), anti-OX40, PD-L1 monoclonal antibody (Anti- B7-H1; MEDI4736), MK-3475 (PD-1 blocker), nivolumab (anti-PD-1 antibody), CT-011 (anti-PD- 1 antibody), BY55 monoclonal antibody, AMP224 (anti-PD-Ll antibody), BMS-936559 (anti-PD- LI antibody), MPLDL3280A (anti-PD-Ll antibody), MSB0010718C (anti-PD-Ll antibody) and ipilimumab (anti-CTLA-4 antibody, also known as Yervoy®, MDX-010 and MDX-101). Exemplary of immunomodulatory antibodies include, but are not limited to, Daclizumab (Zenapax), Bevacizumab (Avastin ®), Basiliximab, Ipilimumab, Nivolumab, pembrolizumab, MPDL3280A, Pidilizumab (CT-011), MK-3475, BMS-936559, MPDL3280A (Atezolizumab), tremelimumab, IMP321, BMS-986016, LAG525, urelumab, PF- 05082566, TRX518, MK-4166, dacetuzumab (SGN-40), lucatumumab (HCD122). SEA-CD40, CP-870, CP-893. MEDI6469, MEDI6383. MOXR0916, AMP-224. MSB0010718C (Avelumab), MEDI4736, PDR001, rHIgM12B7, Ulocuplumab, BKT140, Varlilumab (CDX- 1127), ARGX-110, MGA271, lirilumab (BMS-986015, IPH2101), IPH2201, ARGX-115, Emactuzumab, CC-90002 and MNRP1685A or an antibody- binding fragment thereof. Other exemplary immunomodulators include, e.g.. afutuzumab (available from Roche®): pegfilgrastim (Neulasta®); lenalidomide (CC-5013, Revlimid®); thalidomide (Thalomid®), actimid (CC4047); and IRX-2 (mixture of human cytokines including interleukin 1, interleukin 2, and interferon gamma, CAS 951209-71-5, available from IRX Therapeutics).

[0177] Programmed cell death 1 (PD-1) is an immune checkpoint protein that is expressed in B cells, NK cells, and T cells (Shinohara et al., 1995, Genomics 23:704-6; Blank et al., 2007, Cancer Immunol Immunother 56:739-45; Finger et al., 1997, Gene 197: 177-87; Pardoll (2012) Nature Reviews Cancer 12:252-264). The major role of PD-1 is to limit the activity of T cells in peripheral tissues during inflammation in response to infection, as well as to limit autoimmunity. PD-1 expression is induced in activated T cells and binding of PD-1 to one of its endogenous ligands acts to inhibit T-cell activation by inhibiting stimulatory kinases. PD-1 also acts to inhibit the TCR “stop signal”. PD-1 is highly expressed on Treg cells and may increase their proliferation in the presence of ligand (Pardoll (2012) Nature Reviews Cancer 12:252-264). Anti-PD 1 antibodies have been used for treatment of melanoma, non-small-celllung cancer, bladder cancer, prostate cancer, colorectal cancer, head and neck cancer, triplenegative breast cancer, leukemia, lymphoma and renal cell cancer (Topalian et al., 2012, N Engl J Med 366:2443-54; Lipson et al., 2013, Clin Cancer Res 19:462-8; Berger et al., 2008, Clin Cancer Res 14:3044-51; Gildener-Leapman et al., 2013, Oral Oncol 49: 1089-96; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85). Exemplary anti- PD-1 antibodies include nivolumab (Opdivo by BMS), pembrolizumab (Keytruda by Merck), pidilizumab (CT-011 by Cure Tech), lambrolizumab (MK-3475 by Merck), and AMP-224 (Merck), nivolumab (also referred to as Opdivo, BMS-936558 or MDX1106; Bristol-Myers Squibb) is a fully human IgG4 monoclonal antibody which specifically blocks PD-1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD-1 are described in US 8.008,449 and W02006 / 121168. Pidilizumab (CT-011; Cure Tech) is a humanized IgGlk monoclonal antibody that binds to PD-1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are described in W02009 / 101611. Pembrolizumab (formerly known as lambrolizumab, and also referred to as Keytruda, MK03475; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are described in US 8,354,509 and W02009 / 114335. Other anti-PD-1 antibodies include AMP 514 (Amplimmune), among others, e.g., anti-PD-1 antibodies described in US 8,609,089, US 2010028330, US 20120114649 and / or US 20150210769. AMP-224 (B7-DCIg; Amplimmune; e.g., described in W02010 / 027827 and WO2011 / 066342), is a PD-L2 Fc fusion soluble receptor that blocks the interaction between PD-1 and B7-H1 .

[0178] PD-L1 (also known as CD274 and B7-H1) and PD-L2 (also known as CD273 and B7- DC) are ligands for PD-1, found on activated T cells. B cells, myeloid cells, macrophages, and some types of tumor cells. Anti-tumor therapies have focused on anti-PD-Ll antibodies. The complex of PD-1 and PD-L1 inhibits proliferation of CD8+ T cells and reduces the immune response (Topalian et al., 2012, N Engl J Med 366:2443-54; Brahmer et al., 2012, N Eng J Med 366:2455-65). Anti- PD-L1 antibodies have been used for treatment of non-small cell lung cancer, melanoma, colorectal cancer, renal-cell cancer, pancreatic cancer, gastric cancer, ovarian cancer, breast cancer, and hematologic malignancies (Brahmer et al.. 2012, N Eng J Med 366:2455-65; Ott et al., 2013, Clin Cancer Res 19:5300-9; Radvanyi et al., 2013, Clin Cancer Res 19:5541; Menzies & Long, 2013, Ther Adv Med Oncol 5:278-85; Berger et al., 2008, Clin Cancer Res 14: 13044-51). Exemplary' anti- PD-L1 antibodies include MDX- 1105 (Medarex). MEDI4736 (Medimmune) MPDL3280A (Genentech), BMS-935559 (Bristol-Myers Squibb) and MSB0010718C. MEDI4736 (Medimmune) is a human monoclonal antibody that binds to PD-L1, and inhibits interaction of the ligand with PD-1. MDPL3280A (Genentech / Roche) is a human Fc optimized IgGl monoclonal antibody that binds to PD-L1. MDPL3280A and other human monoclonal antibodies to PD-L1 are described in U.S. Patent No. 7,943,743 and U.S. Publication No. 20120039906. Other anti-PD-Ll binding agents include YW243.55.S70 (see WO2010 / 077634) and MDX-1105 (also referred to as BMS- 936559, and, e.g., anti-PD-Ll binding agents described in W02007 / 005874).

[0179] Also provided herein are methods for treating or delaying the development or progression of cancer in an individual, comprising administering to the subject an effective amount of an additional therapeutic agent, such as an immune checkpoint inhibitor, and a nanoparticle or a composition comprising the same.

[0180] The additional therapeutic agent and the nanoparticle may be administered in any order. For example, an additional therapeutic agent and a nanoparticle may be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, an additional therapeutic agent and a nanoparticle are in separate compositions. In some embodiments, an additional therapeutic agent and a nanoparticle are in the same composition.

[0181] In some embodiments, prior to treatment with an additional therapeutic agent and nanoparticles in accordance with any of the methods described herein, the individual has progressed after treatment with or failed to respond adequately to treatment with a monotherapy of the additional therapeutic agent, e.g., treatment with an immune checkpoint inhibitor, e.g., anti-PD-1 antibody.

[0182] The additional therapeutic agent and the nanoparticle may be administered by the same route of administration or by different routes of administration. In some embodiments, the additional therapeutic agent is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally.

[0183] In some embodiments, the methods may further comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzymaticinhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side- effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation.

[0184] In some aspects, the nanoparticles or compositions comprising the same, are administered together with an adjuvant. Exemplary adjuvants include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) microparticles (Shah et al.. Methods Mol Biol, 1494: 1-14, 2017). In some embodiments, the compositions further comprises an aluminum salt adjuvant to which the nucleic acid encoding the exogenous molecule, such as vaccine epitopes, is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxy phosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant consists of aluminum hydroxide. Other exemplary adjuvants include, but are not limited to. squalene-in-water emulsion (e.g., MF59 or AS03), and / or a saponin such as Quil A or QS-21. as in AS01 or AS02), and resiquimod.IV. DEFINITIONS

[0185] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0186] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matterpertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.

[0187] As used herein, the singular forms “a,” “an," and “the" include plural referents unless the context clearly dictates otherwise. For example, “a" or “an" means “at least one” or “one or more.”

[0188] The term “about” as used herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[0189] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0190] The terms “nucleic acid” and “nucleotide” include naturally-occurring species or functional analogs thereof, or variants thereof. A nucleic acid can contain nucleotides having any of a variety of analogs of these sugar moieties that are established in the field. A nucleic acid can include native or non-native nucleosides. In this regard, a native ribonucleic acid (RNA) can have one or more nucleosides selected from the group consisting of uridine (U), adenosine (A), cytidine (C), or guanosine (G). Useful non-native nucleosides are established in the field, such as a pseudouridine in place of a uridine.

[0191] In some embodiments, “treatment” (and grammatical variations thereof such as “treat” or “treating”) refers to complete or partial amelioration or reduction of a disease or condition or disorder, or a symptom, adverse effect or outcome, or phenotype associated therewith. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. The terms do not imply complete curing of a disease or complete elimination of any symptom or effect(s) on all symptoms or outcomes.

[0192] In some embodiments, “preventing,” as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of a disease in a subject that may be predisposed to the disease but has not yet been diagnosed with the disease. In some embodiments, the provided molecules and compositions are used to delay development of a disease or to slow the progression of a disease. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, the subject is a human.

[0193] A “therapeutically effective amount” of an agent, e.g.. a pharmaceutical formulation, binding molecule, or cells, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease, condition, or disorder, and / or pharmacokinetic or pharmacodynamic effect of the treatment. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject, and the populations of cells administered. In some embodiments, the provided methods involve administering the fusion protein, nucleic acid constructs, and / or compositions at effective amounts, e.g., therapeutically effective amounts.

[0194] A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0195] As used herein, a “subject” is a mammal, such as a human or other animal, and tj pically is human.

[0196] As used herein, “delaying development of a disease” means to defer, hinder, slow, retard, stabilize, suppress and / or postpone development of the disease (such as cancer). This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. For example, a late stage cancer, such as development of metastasis, may be delayed.

[0197] The term “peptide” as used herein refers to a molecule comprising an amino acid sequence of between 2 and 200 amino acids, connected by peptide bonds, but which can comprise non-amino acid structures. Peptides according to the invention can contain any of the conventional 20 amino acids or modified versions thereof, or can contain non-naturally occurring amino-acids incorporated by chemical peptide synthesis or by chemical or enzy matic modification.

[0198] The term “antigen” as used herein refers to a structure of a macromolecule, typically protein (with or without polysaccharides) or made of proteinaceous composition and comprising T cell or B cell epitopes.

[0199] The term “epitope” refers to one or several portions (which may define a conformational epitope) of an antigenic protein which is / are specifically recognized and bound by an antibody or a portion thereof or a receptor presented at the cell surface of a B or T cell lymphocyte, and which is able, by said binding, to induce an immune response. The term “T cell epitope” in the context of the present invention refers to a dominant, sub-dominant or minor T cell epitope, i.e., a part of an antigenic protein that is specifically recognized and bound by a receptor at the cell surface of a T lymphocyte. Whether an epitope is dominant, sub-dominant or minor depends on the immune reaction elicited against the epitope. Dominance depends on the frequency at which such epitopes are recognized by T cells and able to activate them, among all the possible T cell epitopes of a protein.

[0200] In some aspects, a T cell epitope is an epitope recognized by MHC class II molecules, w hich typically includes about 8-10 amino acids which fit in the groove of the MHC II molecule. Peptides recognized by MHC class II molecules and not by MHC class I molecules are referred to as MHC class II restricted T cell epitopes.

[0201] The term “MHC” refers to “major histocompatibility antigen”. In humans, the MHC genes are known as HLA (“human leukocyte antigen”) genes. Although there is no consistently followed convention, some literature uses HLA to refer to HLA protein molecules, and MHC to refer to the genes encoding the HLA proteins. As such the terms “MHC” and “HLA” are equivalents when used herein.

[0202] The term “sequence identity” of two sequences as used herein relates to the number of positions with identical nucleotides or amino acids divided by the number of nucleotides or amino acids in the shorter of the sequences, when the two sequences are aligned. In particular, the sequence identity is from 70% to 80%, from 81% to 85%, from 86% to 90%, from 91% to 95%, from 96% to 100%, or 100%.

[0203] The terms “peptide-encoding nucleic acid” and “nucleic acid encoding peptide” as used herein refer to a nucleotide sequence, which, when expressed in an appropriate environment, results in the generation of the relevant peptide sequence or a derivative or homologue thereof. Such polynucleotides or nucleic acids include the normal sequences encoding the peptide, as well as derivatives and fragments of these nucleic acids capable of expressing a peptide with the required activity. The nucleic acid encoding a peptide according to the invention or fragment thereof is a sequence encoding the peptide or fragment thereof originating from a mammal or corresponding to a mammalian, most particularly a human peptide fragment.

[0204] The term “immune disorders” or “immune diseases” refers to diseases wherein a reaction of the immune system is responsible for or sustains a malfunction or non-physiological situation in an organism. Included in immune disorders are, inter alia, allergic disorders and autoimmune diseases.

[0205] The terms “allergic diseases” or “allergic disorders” as used herein refer to diseases characterized by hypersensitivity reactions of the immune system to specific substances called allergens (such as pollen, stings, drugs, or food). Allergy is the ensemble of signs and symptoms observed whenever an atopic individual patient encounters an allergen to which he has been sensitized, which may result in the development of various diseases, in particular respiratory diseases and symptoms such as bronchial asthma. Various types of classifications exist and mostly allergic disorders have different names depending upon where in the mammalian body it occurs. “Hypersensitivity” is an undesirable (damaging, discomfortproducing and sometimes fatal) reaction produced in an individual upon exposure to an antigento which it has become sensitized; “immediate hypersensitivity’" depends of the production of IgE antibodies and is therefore equivalent to allergy.

[0206] The term “natural” when referring to a peptide relates to the fact that the sequence is identical to a fragment of a naturally occurring protein (wild type or mutant). In contrast, the term “artificial" refers to a sequence which as such does not occur in nature. An artificial sequence is obtained from a natural sequence by limited modifications such as changing / deleting / inserting one or more amino acids within the naturally occurring sequence or by adding / removing amino acids N- or C-terminally of a naturally occurring sequence.V. EXEMPLARY EMBODIMENTS

[0207] Among the provided embodiments are:1. A nanoparticle comprising:(a) N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA);(b) 1.2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and(c) a galactosylceramide.2. The nanoparticle of embodiment 1, wherein the galactosylceramide is alphagalactosylceramide (a-GalCer).3. The nanoparticle of embodiment 1 or 2, wherein the nanoparticle further comprises a nucleic acid.4. The nanoparticle of embodiment 3, wherein the nucleic acid comprises a ribonucleic acid (RNA).5. The nanoparticle of embodiment 3 or 4, wherein the nucleic acid comprises a circular RNA.6. The nanoparticle of embodiment 3 or 4, wherein the nucleic acid comprises a linear RNA.7. The nanoparticle of any of embodiments 3, 4, and 6, wherein the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2', 3'- cychc phosphate at the 3' terminus.8. The nanoparticle of any of embodiments 3, 4, and 6, wherein the nucleic acid comprises a linear messenger RNA (mRNA).9. The nanoparticle of any of embodiments 3, 4, and 6, wherein the nucleic acid comprises a micro RNA (miRNA).10. The nanoparticle of any of embodiments 3, 4, and 6. wherein the nucleic acid comprises a small inhibitory RNA (siRNA).11. The nanoparticle of any of embodiments 3, 4, and 6, wherein the nucleic acid comprises a guide RNA (gRNA).12. The nanoparticle of any of embodiments 3-11. wherein the nanoparticle forms a lipoplex (LPX).13. The nanoparticle of any of embodiments 1-12, wherein the ratio of DOTMA:DOPE is between about 5: 1 and about 1:5.14. The nanoparticle of any of embodiments 1-13, wherein the ratio of DOTMA:DOPE is between about 3: 1 and about 1:3.15. The nanoparticle of any of embodiments 1-14, wherein the ratio of DOTMA:DOPE is about 2: 1.16. The nanoparticle of any of embodiments 1-15, wherein the proportion of a-GalCer is between about 0.1% and about 5% molar percentage.17. The nanoparticle of any of embodiments 1-16. wherein the proportion of a-GalCer is between about 0.5% and about 2% molar percentage.18. The nanoparticle of any of embodiments 1-17, wherein the proportion of a-GalCer is about 0.5% molar percentage.19. The nanoparticle of any of embodiments 1-17. wherein the proportion of a-GalCer is about 1 % molar percentage.20. The nanoparticle of any of embodiments 1-17, wherein the proportion of a-GalCer is about 2% molar percentage.21. The nanoparticle of any of embodiments 3-20. wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is between about 1:2 to about 2: 1.22. The nanoparticle of any of embodiments 3-21, wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is about 1.3:2.23. The nanoparticle of any of embodiments 1-22. wherein the nucleic acid comprises a nucleic acid sequence encoding one or more exogenous molecules.24. The nanoparticle of embodiment 23, wherein the one or more exogenous molecules is selected from among a vaccine epitope, a cancer epitope, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.25. The nanoparticle of embodiment 23 or 24. wherein the one or more exogenous molecule comprises a vaccine epitope.26. The nanoparticle of embodiment 25, wherein the vaccine epitope comprises an infectious disease vaccine epitope, optionally a viral vaccine epitope.27. The nanoparticle of embodiment 25 or 26, wherein the vaccine epitope comprises a cancer epitope.28. The nanoparticle of embodiment 27, wherein the cancer epitope is a cancer neoantigen epitope.29. The nanoparticle of embodiment 27 or 28, wherein the cancer neoantigen epitope comprises a cancer epitope selected from among ME-1. M26. Gludl. Mtchl. Ml 8. and E2f8.30. The nanoparticle of embodiment 23 or 24, wherein the one or more exogenous molecules comprises a sequence-specific nuclease.31. The nanoparticle of embodiment 30, wherein the sequence-specific nuclease is a Cas nuclease.32. The nanoparticle of embodiment 31. wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Cas 12 nuclease, or a Cas 13 nuclease.33. The nanoparticle of embodiment 23 or 24, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.34. The nanoparticle of embodiment 23 or 24. wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.35. The nanoparticle of embodiment 34, wherein the immunomodulatory polypeptide comprises a cytokine.36. The nanoparticle of embodiment 23 or 24, wherein the one or more exogenous molecules comprises a transcription factor.37. The nanoparticle of embodiment 23 or 24, wherein the one or more exogenous molecules comprises a reporter molecule.38. The nanoparticle of embodiment 37, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP)39. The nanoparticle of any of embodiments 1-38, wherein when the nanoparticle is administered to a subject, an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle.40. The nanoparticle of embodiment 39, wherein the immune response comprises an increase in cytokine production.41. The nanoparticle of embodiment 40, wherein the cytokine is selected from among one or more of: IFN-y, IL-2, IL-6, and IL-12.42. The nanoparticle of embodiment 39, wherein the immune response comprises an increase in co-stimulatory molecule expression on dendritic cells.43. The nanoparticle of embodiment 42, wherein the co-stimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.44. The nanoparticle of embodiment 39, wherein the immune response comprises activation of immune cells.45. The nanoparticle of embodiment 44, wherein the immune cell is selected from among one or more of: NKT, NK, CD4+ T, and CD8+ T cells.46. The nanoparticle of any of embodiments 39-45, wherein the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity of tumor-specific T cells, and increase in migration of tumor-specific T cells.47. The nanoparticle of any of embodiments 39-46, wherein the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.48. The nanoparticle of any of embodiments 1-47. wherein when administered to a subject, the nanoparticle is localized to the spleen.49. The nanoparticle of any of embodiments 1-48, wherein when administered to a subject, the nanoparticle is localized to a tumor.50. The nanoparticle of any of embodiments 1-49. wherein when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node.51. The nanoparticle of any of embodiments 1-50, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen.52. The nanoparticle of any of embodiments 1-51. wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor.53. The nanoparticle of any of embodiments 1-52, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor-draining lymph node.54. The nanoparticle of any of embodiments 1-53. wherein when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor grow th, or increasing survival of the subject.55. A method for generating a nanoparticle, the method comprising generating a lipid mixture of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and incorporating an a galactosylceramide to the mixture.56. The method of embodiment 55, wherein the galactosylceramide is alphagalactosylceramide (a-GalCer).57. The method of embodiment 55 or 56, wherein The method forms a lipoplex (LPX).58. The method of any of embodiments 55-57, wherein the ratio of DOTMA:DOPE is between about 5:1 and about 1:5.59. The method of any of embodiments 55-58, wherein the ratio of DOTMA:DOPE is between about 3: 1 and about 1:3.60. The method of any of embodiments 55-59, wherein the ratio of DOTMA:DOPE is about 2: 1.61. The method of any of embodiments 55-60, w herein the proportion of a-GalCer is between about 0.1% and about 5% molar percentage.62. The method of any of embodiments 55-61, w herein the proportion of a-GalCer is between about 0.5% and about 2% molar percentage.63. The method of any of embodiments 55-62, wherein the proportion of a-GalCer is about 0.5% molar percentage.64. The method of any of embodiments 55-62, wherein the proportion of a-GalCer is about 1 % molar percentage.65. The method of any of embodiments 55-62, wherein the proportion of a-GalCer is about 2% molar percentage.66. The method of any of embodiments 55-65, further comprising mixing the nanoparticle with a nucleic acid.67. The method of embodiment 66, wherein the nucleic acid comprises a ribonucleic acid (RNA).68. The method of embodiment 65 or 66, wherein the nucleic acid comprises a circularRNA.69. The method of embodiment 65 or 66, wherein the nucleic acid comprises a linearRNA.70. The method of any of embodiments 65, 66, and 69, wherein the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2', 3'- cyclic phosphate at the 3' terminus.71. The method of any of embodiments 65, 66, and 69, wherein the nucleic acid comprises a linear messenger RNA (mRNA).72. The method of any of embodiments 65, 66, and 69, wherein the nucleic acid comprises a micro RNA (miRNA).73. The method of any of embodiments 65, 66, and 69, wherein the nucleic acid comprises a small inhibitory RNA (siRNA).74. The method of any of embodiments 65, 66, and 69, wherein the nucleic acid comprises a guide RNA (gRNA).75. The method of any of embodiments 66-74, wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is between about 1:2 to about 2: 1.76. The method of any of embodiments 66-75, wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is about 1.3:2.77. A nanoparticle generated by the method of any of embodiments 55-76.78. A composition comprising the nanoparticle of any of embodiments 1-54 and 77.79. The composition of embodiment 78, wherein the composition is a pharmaceutical composition.80. The composition of embodiment 78 or 79, wherein the composition comprises a pharmaceutically acceptable excipient.81. A method of vaccinating a subject, the method comprising administering the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 to a subject.82. A method for enhancing an immune response to a vaccine epitope in a subject, the method comprising administering the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 to a subject.83. A method of treating a disease or disorder in a subject, the method comprising administering the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 to a subject.84. The method of any of embodiments 81-83, wherein an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle or the composition.85. The method of embodiment 84, wherein the immune response comprises an increase in cytokine production.86. The method of embodiment 85, wherein the cytokine comprises one or more of: IFN-y, IL-2, IL-6, and IL- 12.87. The method of embodiment 85 or 86, wherein the cytokine comprises IFN-y.88. The method of any of embodiments 84-87, wherein the immune response comprises an increase in co-stimulatory molecule expression on dendritic cells.89. The method of embodiment 88, wherein the immune response comprises an increase in co-stimulatory molecule expression on MHC-II CDl lb CDl lc+dendritic cells.90. The method of embodiment 88, wherein the immune response comprises an increase in co-stimulatory molecule expression on MHC-II+CDl lb+CDl lc+dendritic cells.91. The method of any of embodiments 88-90, wherein the co-stimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.92. The method of any of embodiments 84-91, wherein the immune response comprises activation of an immune cell.93. The method of embodiment 92, wherein the immune cell is selected from among one or more of: NKT, NK, CD4+ T, and CD8+ T cells.94. The method of any of embodiments 84-91, wherein the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity of tumor-specific T cells, and increase in migration of tumor-specific T cells.95. The method of any of embodiments 84-94, wherein the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.96. The method of any of embodiments 81-95, wherein when administered to a subject, the nanoparticle is localized to the spleen.97. The method of any of embodiments 81-96, wherein when administered to a subject, the nanoparticle is localized to a tumor.98. The method of any of embodiments 81-97, wherein when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node.99. The method of any of embodiments 81-98, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen.100. The method of any of embodiments 81-99, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor.101. The method of any of embodiments 81-100, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor-draining lymph node.102. The method of any of embodiments 81-101, wherein when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor growth, or increasing survival of the subject.103. The method of any of embodiments 81-102, wherein the method prevents or reduces the severity of a disease or disorder associated with the vaccine epitope in the subject.104. The method of embodiment 103, wherein the disease or disorder is an infectious disease, optionally a viral infection.105. The method of embodiment 103 or 104, wherein the disease or disorder is a cancer.106. The method of any of embodiments 81-105, wherein the administration is by intravenous administration.107. The nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 for use in vaccinating a subject, wherein the nanoparticle, or the composition is administered to the subject.108. The nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 for use in enhancing an immune response to a vaccine epitope in a subject, wherein the nanoparticle, or the composition is administered to the subject.109. The nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 for use in treating a disease or disorder in a subject, wherein the nanoparticle, or the composition is administered to the subject.110. Use of the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 in the manufacture of a medicament for vaccinating a subject, wherein the medicament is administered to the subject.111. Use of the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 in the manufacture of a medicament for enhancing an immuneresponse to a vaccine epitope in a subject, wherein the medicament is administered to the subject.112. Use of the nanoparticle of any of embodiments 1-54 and 77, or the composition of any of embodiments 78-80 in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.VI. EXAMPLES

[0208] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1; Generation and characterization of a-Galactosylceramide-containing lipid nanoparticles

[0209] Various concentrations of alpha-galactosylceramide (a-GalCer), a natural ligand for Natural killer T (NKT) cells, were incorporated into lipoplex (LPX) nanoparticles comprising the lipids l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2- dioleoyl-sn-glycero- 3 -phosphoethanolamine (DOPE), and complexed with an exemplary circular RNA molecule (referred to as LPX / a-GalCer / circular RNA), for characterization.

[0210] Briefly, to make LPX / a-GalCer / circular RNA nanoparticles, lipid mixtures composed of DOTMA and DOPE were prepared at 2: 1 molar ratio. a-GalCer was incorporated into the lipid mixture at the total lipid molar percentage, 0.5%, 1.0%, or 2.0%. After determining the DOTMA concentration, LPX / a-GalCer / circular RNA nanoparticles were assembled with a 1.3:2 charge ratio of liposome (DOTMA) to RNA by using the NanoAssemblr Platform (Precision Nanosystems Inc., Vancouver, BC. Canada). Circular RNA- containing LPX nanoparticles comprising DOTMA / DOPE without a-GalCer (LPX / circular RNA) were also assessed.

[0211] The LPX / a-GalCer / circular RNA nanoparticles were characterized for physical properties, such as size (FIG. IB), poly dispersity index (PDL FIG. 1C) and zeta potential (FIG. ID). The size of LPX / circular RNA without a-GalCer was 295.9 ±1.5. The size of LPX / circular RNA containing 0.5% (311.6 ±2.0), 1.0% (300.6 ±3.0), and 2.0% (329.7 ±0.6) of a-GalCer was bigger than that of LPX / circular RNA without a-GalCer (FIG. IB).

[0212] PDI is an indicator of the colloidal solution of nanoparticles that describes the width or spread of the particle size distribution. Nanoparticles accumulate into clusters due to agglomeration at the high PDI values > 0.7. As shown in FIG. 1C, PDI value of theLPX / circular RNA without a- GalCer or with 0.5%. 1.0%, or 2.0% of a-GalCer was below 0.2, indicating a low propensity to accumulate into clusters for the LPX / circular RNA, both with and without a-GalCer.

[0213] Zeta potential is a measure of the effective electric charge on the nanoparticle’s surface, quantifying the charges. When a nanoparticle has a net surface charge, the charge is screened by the concentration of ions of opposite charge near the nanoparticle surface. The zeta potential of LPX / circular RNA without a-GalCer was -29.37 ±0.58. The zeta potential of LPX / circular RNA containing 0.5% (-26.37 ±1.53), 1.05 (-29.97±1.09), 2.0% (-29.40 ±0.1.64) of a-GalCer was similar to that of LPX / circular RNA without a-GalCer (FIG. ID) indicating a- GalCer did not change the surface conditions of the nanoparticles. The results show that the general characteristics of a- GalCer containing LPX nanoparticles were similar to those without a-GalCer.Example 2: Delivery and in vivo localization of DOTMA / DOPE lipid nanoparticles

[0214] Lipid nanoparticles containing a circular RNA were administered to mice to assess the delivery and in vivo organ and cellular localization of the nanoparticles.

[0215] To identify the organ to which LPX is delivered, a DOTMA / DOPE LPX containing a circular RNA encoding firefly luciferase was generated generally as described in Example 1. and luciferase activity was examined 6 hours after intravenous (IV) injection of LPX / circular RNA, or a control without LPX / circular RNA into Balb / c mice.

[0216] As shown in FIG. 2A, luciferase activity was observed in the spleen region of the mice. To confirm that the spleen is the primary organ to which LPX / RNA is delivered, the spleen, the lungs, the liver and the large intestine were isolated from a mouse at 24 and 48 hours after IV injection, and firefly luciferase activity was examined. As shown in FIG. 2A and FIG. 2B, firefly luciferase activity was observed only in the spleen and not in the lungs, the liver, or the large intestine. These results demonstrate that the LPX / circular RNA was selectively- delivered to the spleen.

[0217] To identify the immune cell subsets that take up the LPX in the spleen, mice were intravenously injected an LPX / mRNA, containing an mRNA encoding an enhanced green fluorescent protein (eGFP). 6 hours after injection, splenocytes were isolated and analyzed for immune cell subset showing eGFP signal by flow cytometry (FIG. 3A). Compared to control, the mean fluorescence intensity (MFI) of the eGFP for dendritic cells (DC) (MHC-II CD11c )was significantly higher while the eGFR MFI for macrophages. CD4 and CD8+T cells, B cells, and natural killer (NK) cells were not (FIGS. 3B-3H). eGFP MFI of neutrophils (CDl lb+Ly49G+) was also significantly higher compared to controls, although at lower levels compared to DCs. These results indicate that LPX / RNA nanoparticles are primarily delivered to DCs in the spleen.Example 3;Immune adjuvant effects of «-Galactosylceramide-containing lipid nanoparticles in cytokine secretion

[0218] The immune adjuvant effects of a-Galactosylceramide-containing lipid nanoparticles with RNA. including the effects on cytokine secretion, were assessed.

[0219] As depicted in FIG. 4, when LPX / a-GalCer / circular RNA nanoparticles delivered to dendritic cells (DCs), a-GalCer can be presented on the cell surface by CDld, which can be recognized by T cell receptors on natural killer T cells (NKT) cells. During this process, NKT cells are activated and secrete various cytokine including interferon gamma (IFN- y), which subsequently activates various immune cells including DCs, macrophages, T cells, and NK cells.

[0220] To assess whether LPX / a-GalCer / circular RNA nanoparticles were delivered to DCs, and whether NKT cells secrete various cytokines upon a-GalCer-mediated stimulation, LPX / a- GalCer / circular RNA nanoparticles were assessed for their effect on cytokine secretion in vivo. LPX / a-GalCer / circular RNA nanoparticles were generated, generally as described in Example 1 above. BALB / c mice were intravenously injected with LPX / circular RNA without a-GalCer (LPX) or LPX containing different amount of a-GalCer (0.5%, 1.0%, or 2.0%) or 2 pg free a-GalCer (not incorporated into LPX) as a positive control (n=5 mice for each group) (FIG. 5A). 6 hours after injection, IFN-y, IL-2, IL-6, and IL- 12 were measured in the mouse serum of each group using enzyme-linked immunosorbent assays (ELISA) or magnetic cytokine multiplex assay kits.

[0221] As shown in FIG. 5B (IFN-y), FIG. 5C (IL-2), FIG. 5D (IL-6), and FIG. 5E (IL- 12), compared to negative control, LPX / circular RNA nanoparticle incorporated with different amounts of a-GalCer (0.5%, 1.0%, or 2.0%) significantly induced secretion of IFN-y, IL-2, IL-6, and IL-12 while LPX / circular RNA nanoparticle without a-GalCer (LPX) did not, generally in an a-GalCer dose-dependent manner. LPX / circular RNA nanoparticles with different amounts of a-GalCer (0.5%, 1.0%. or 2.0%) secreted relatively lower levels of IL-2,IL-6, and IL-12 compared to free a- GalCer (see FIG. 5C, FIG. 5D, and FIG. 5E, respectively). However, LPX / circular RNA nanoparticles containing 1.0% and 2.0% a-GalCer secreted similar levels of IFN-y as free a-GalCer (see FIG. 5B).

[0222] These results indicate that unlike LPX alone, a-Galactosylceramide-containing lipid nanoparticles exhibits an immune adjuvant function and is able to induce the production of cytokines that activate immune cells.Example 4;The role of delivery route of a-Galactosylceramide-containing lipid nanoparticles in inducing immune cell activation

[0223] Whether the route of delivery influences the stimulation of immune cells by a- Galactosylceramide-containing lipid nanoparticles was assessed.

[0224] LPX / a-GalCer / circular RNA nanoparticles with 2% a-GalCer and LPX / circular RNA nanoparticles (without a-GalCer) were generated, generally as described in Example 1 above, and administered to mice using different delivery routes: by injecting mice via intravenous (IV) or intramuscular (IM) routes. 3 days after injection, splenocytes were isolated and analyzed for the activation status of immune cells (FIG. 6A) by flow- cytometry. Lymphocytes were stained for DAPI, CD45, CD3, CD4, CD8, CD69, CD49b, and gated for NKTs (CD45 CD3 CD49b ), NK cells (CD45+CD3 CD49b+), CD4+T cells (CD45 CD3 CD4 CD8 ), and CD8+T cells (CD45+CD3+CD4 CD8 ). Antigen-presenting cells (APC) were stained for DAPI, CD45, CDl lb, CDl lc, H-2Kd, MHCII, CD40, CD80, CD86, and gated for macrophages (CD45+CDllb+MCHII+CDl lc ), CDl lb+DC (CD45 MHCII CD1 Ib CDllc ), dendritic cells (CD45+MHCII+CDl lb CDl lc+), and B cells (CD45+MHCII+CDl lb‘CDl lc ). Two subsets of DCs (MHC-II+CDl lb’CDl lc+and MHC- II+CD1 lb+CDl lc+) were analyzed for expression of co- stimulatory molecules including CD40, CD80, and CD86.

[0225] As shown in FIGS. 6B-6D, for the MHC-II CD1 lb’CDl lc+DC subset, neither LPX / circular RNA nor LPX / a-GalCer / circular RNA had any effect on the expression of CD40 molecules, regardless of the IV or IM delivery route (FIG. 6B). When LPX / a-GalCer / circular RNA was administered via IV rather than IM, CD80 and CD86 were significantly upregulated in the MHC-II CDl lb CDl lc+DC subset (see FIG. 6C and FIG. 6D, respectively). Furthermore, LPX / circular RNA had no effect on CD80 and CD86 expression by either the IV or the IM delivery route (FIG. 6C and 6D).

[0226] As shown in FIGS. 6E-6G, for the MHC-II CD1 Ib CDl lc+DC subset, neither LPX / circular RNA nor LPX / a-GalCer / circular RNA had any effect on the expression of CD80 molecules when compared to control, regardless of the IV or IM delivery route (FIG. 6F). However, when LPX / a-GalCer / circular RNA was administered via IV rather than IM, CD40 and CD86 were significantly upregulated (see FIG. 6E and FIG. 6G. respectively). Furthermore. LPX / circular RNA had no effect on CD40 and CD86 expression by either the IV or the IM delivery route see FIG. 6E and FIG. 6G, respectively).

[0227] As co-stimulatory molecules such as CD40, CD80 and CD86 expressed by DCs play an important role in the activation of T cells, these results show that LPX / a- GalCer / circular RNA can induce activation of two subsets of DCs when injected via IV rather than IM. On the other hand, LPX / circular RNA did not affect the expression of these costimulatory molecules on DCs regardless of the IV and IM delivery routes.

[0228] A similar result was observed in the activation of lymphocytes: when the expression of CD69, which indicates lymphocyte activation, was assessed, LPX / a- GalCer / circular RNA significantly increased CD69 expression on NKT, NK. CD4+T and CD8+T cells only by the IV administration, but not by IM (FIG. 6H, FIG. 61, FIG. 6J, and FIG. 6K, respectively). However, LPX / circular RNA had no effect on the activation of these lymphocytes regardless of the IV or the IM delivery routes.

[0229] The results indicate that a-Galactosylceramide-containing lipid nanoparticles can induce lymphocyte activation when administered via the IV rather than the IM route while LPX / circular RNA does not affect lymphocyte activation regardless of the delivery' route.Example 5;Targeted delivery of <z-Galactosylceramide-containing lipid nanoparticles to the tumor microenvironment via IV administration

[0230] Whether a-Galactosylceramide-containing lipid nanoparticles can be targeted to tumors and tumor-draining lymph nodes was assessed in a mouse tumor model.

[0231] LPX / a-GalCer / mRNA (encoding firefly luciferase) nanoparticles with 2% (j- GalCer (LPX / a-GalCer (2%) / mRNA) were generated, generally as described in Example 1 above. The nanoparticles were injected into tumor-bearing mice via IV and IM delivery routes (FIG. 7A). Spleens, tumors, tumor draining lymph nodes, and non-tumor lymph nodes were collected the day after administration of the LPX / a-GalCer (2%) / mRNA and analyzed for firefly luciferase activity (FIG. 7A and FIG. 7B).

[0232] Compared to control, firefly luciferase activity was significantly higher in the spleen of only the mice administered the LPX / a-GalCer (2%) / mRNA nanoparticles via IV, but not via IM (FIG. 7C). Significantly higher firefly luciferase activity than controls was also observed in the tumor (FIG. 7D) and inguinal lymph nodes (FIG. 7E; which are tumordraining lymph nodes (TDLN) in this mouse model), only in mice administered the LPX / a- GalCer (2%) / mRNA nanoparticles via IV. but not IM. However, in the axillary (FIG. 7F) and mesenteric lymph nodes (FIG. 7G), which are non-tumor-draining lymph nodes, there were no differences in firefly luciferase activity between IM and IV injections compared to controls. These results indicate that a- Galactosylceramide-containing lipid nanoparticles selectively deliver the mRNA only to the spleen, the tumor, and tumor-draining lymph nodes. These results support that a-Galactosylceramide- containing lipid nanoparticles deliver their RNA payload to the spleen, which may influence systemic immunity, and to the tumor and tumor draining lymph nodes, which may modulate the tumor microenvironment. These observations indicate that a-Galactosylceramide-containing lipid nanoparticles can be developed as an effective way to control tumors by selectively targeting and delivering RNA drugs, such as therapeutic mRNAs, circular RNAs, siRNAs and microRNAs, to the tumor microenvironment, and can assist in converting the immunosuppressive environment commonly associated with tumors to an immune-promoting environment and to support anti-tumor immunity.Example 6;Delivery of cancer neoantigen-encoding nucleic acids by a- Galactosylceramide-containing lipid nanoparticles and anti-cancer effects

[0233] a-Galactosylceramide-containing lipid nanoparticles were used to deliver cancer neoantigen-encoding nucleic acids to tumor bearing mice, and the anti-cancer effects of the neoantigen vaccination was assessed.

[0234] To investigate the anti-cancer effects of a cancer neoepitope vaccine, LPX / a- GalCer / circular RNA (encoding various cancer neoantigen epitopes) or LPX / circular RNA (encoding the same cancer neoantigen epitopes) without a-GalCer were generated generally as described in Example 1 above. The nanoparticles were administered via IV, alone or in combination with a PD-1 inhibitor (anti-PD-1 antibody) administered intraperitoneally (IP), to CT26 tumor model, a syngeneic colon cancer cell line of BALB / c mice inoculated into mice (FIG. 8A)

[0235] As shown in FIG. 8B, IV administration of LPX / a-GalCer / circular RNA (LPX / a- GalCer) alone effectively controlled tumor growth, whereas LPX / circular RNA (LPX) did not. Furthermore, this anti-cancer effect of LPX / a-GalCer monotherapy was not significantly different when compared to the PD-1 inhibitor monotherapy, as well as LPX / a- GalCer + PD-1 inhibitor combination therapy or LPX + PD-1 inhibitor combination therapy. As shown in FIG. 8C, a similar result was also observed in the survival rate of mice.

[0236] The clinical success of PD-1 inhibitors has been attributed to their ability to selectively act on tumors and tumor-draining lymph nodes, which increases the efficacy of the drug and reduces side effects. As shown in FIG. 8B and FIG. 8C, administration of nucleic acids encoding cancer neoantigens, in a a-Galactosylceramide-containing lipid nanoparticles was observed to have a comparable anti-cancer effect as a PD-1 inhibitor monotherapy. The results support the utility' of a-Galactosylceramide-containing lipid nanoparticles in delivering anti-cancer agents, including cancer neoantigen vaccine-encoding nucleic acids, to therapeutically relevant sites of the body, such as the tumor or tumor-draining lymph nodes. The results also support the use of Galactosylceramide-containing lipid nanoparticles in delivering various types of nucleic acid payloads, such as RNAs, linear mRNAs, circular RNAs, siRNAs, microRNAs, and others.

[0237] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.

[0238] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.Example 7;LPX-aGalcer nanoparticle (LPXa) as a delivery tool for RNA therapeutics

[0239] To assess LPX-aGalcer nanoparticle (LPXa) as a delivery tool for RNA therapeutics, mRNA encoding eGFP was encapsulated in LPXa, or LPX and injected intravenously into five mice in each group (PBS was used as control). 6 hours after injection, splenocytes were isolated and analyzed for immune cell subset showing eGFP-positive cells by FACS (FIG. 9A). Fluorescence microscopy of eGFP-positive cells confirmed that LPXa was delivered to splenocytes (FIG. 9B). LPXa was found to primarily delivered to conventional dendritic cells (eDCs) and plasmacytoid dendritic cells (pDCs) (FIG. 9C-9D). When conventional dendritic cells (eDCs, Lin CD45+CDl lchlMHC-IIhl), plasmacytoid dendritic cells (pDCs, Lin CD45+CDl lcloMHC-IIhlCD137hi), macrophages (Lin-CD45+F4 / 80+CDl lb+), neutrophils (Lin’CD45+CDl lb+Ly6G+), B cells (CD3 CD19 ), CD4+T. CD8+T cells were analyzed by FACS, compared to the PBS control, LPXa targeted approximately 5% of eDCs and 10% of pDCs, respectively. Notably, LPXa was slightly higher in targeting pDCs than LPX. On the other hand, given the very low percentage of eGFP-positive cells in macrophages, neutrophils, B cells, CD4+T, and CD8+T cells, the targeting efficiency of LPXa on these cells appears to be very low. Thus, these results show that LPXa nanoparticles are primarily delivered to eDCs and pDCs in the spleen. Given that professional antigen-presenting cells such as eDCs and pDCs play a key role in regulating immune responses, including T cell responses, these results also suggest that LPXa may provide a tool for delivery in a variety of RNA therapeutics, including circular RNAs. which regulate the expression of various molecules that modulate immune responses in both anti-tumor immunity and autoimmune diseases.

Claims

Claims1. A nanoparticle comprising:(a) N-[l-(2,3-dioleyloxy)propyl]-N,N.N-trimethylammonium chloride (DOTMA);(b) 1.2-dioleoyl-sn-glycero-3-phosphoethanolamme (DOPE); and(c) a galactosylceramide.

2. The nanoparticle of claim 1, wherein the galactosylceramide is alphagalactosylceramide (a-GalCer).

3. The nanoparticle of claim 1 or 2, wherein the nanoparticle further comprises a nucleic acid.

4. The nanoparticle of claim 3, wherein the nucleic acid comprises a ribonucleic acid (RNA).

5. The nanoparticle of claim 3 or 4, wherein the nucleic acid comprises a circular RNA.

6. The nanoparticle of claim 3 or 4, wherein the nucleic acid comprises a linear RNA.

7. The nanoparticle of any of claims 3. 4, and 6. wherein the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the S' terminus, and a 2',3'-cyclic phosphate at the 3' terminus.

8. The nanoparticle of any of claims 3. 4, and 6. wherein the nucleic acid comprises a linear messenger RNA (mRNA).

9. The nanoparticle of any of claims 3. 4, and 6, wherein the nucleic acid comprises a micro RNA (miRNA).

10. The nanoparticle of any of claims 3, 4, and 6, wherein the nucleic acid comprises a small inhibitory' RNA (siRNA).

11. The nanoparticle of any of claims 3, 4, and 6, wherein the nucleic acid comprises a guide RNA (gRNA).

12. The nanoparticle of any of claims 3-11, wherein the nanoparticle forms a lipoplex(LPX).

13. The nanoparticle of any of claims 1-12, wherein the ratio of DOTMA:DOPE is between about 5:1 and about 1:5.

14. The nanoparticle of any of claims 1-13, wherein the ratio of DOTMA:DOPE is between about 3:1 and about 1:3.

15. The nanoparticle of any of claims 1-14, wherein the ratio of DOTMA:DOPE is about 2: 1.

16. The nanoparticle of any of claims 1-15, wherein the proportion of a-GalCer is between about 0.1% and about 5% molar percentage.

17. The nanoparticle of any of claims 1-16, wherein the proportion of a-GalCer is between about 0.5% and about 2% molar percentage.

18. The nanoparticle of any of claims 1-17, wherein the proportion of a-GalCer is about 0.5% molar percentage.

19. The nanoparticle of any of claims 1-17, wherein the proportion of a-GalCer is about 1 % molar percentage.

20. The nanoparticle of any of claims 1-17, wherein the proportion of a-GalCer is about 2% molar percentage.

21. The nanoparticle of any of claims 3-20, wherein the nucleic acid is a ribonucleic acid (RNA), and the D0TMA:RNA charge ratio is between about 1:2 to about 2:1.

22. The nanoparticle of any of claims 3-21, wherein the nucleic acid is a ribonucleic acid (RNA), and the D0TMA:RNA charge ratio is about 1.3:2.

23. The nanoparticle of any of claims 1-22, wherein the nucleic acid comprises a nucleic acid sequence encoding one or more exogenous molecules.

24. The nanoparticle of claim 23, wherein the one or more exogenous molecules is selected from among a vaccine epitope, a cancer epitope, a nuclease, a guide RNA (gRNA), a therapeutic polypeptide, an antibody or an antigen-binding fragment thereof, an immunomodulatory polypeptide, a transcription factor, or a reporter molecule.

25. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecule comprises a vaccine epitope.

26. The nanoparticle of claim 25, wherein the vaccine epitope comprises an infectious disease vaccine epitope, optionally a viral vaccine epitope.

27. The nanoparticle of claim 25 or 26, wherein the vaccine epitope comprises a cancer epitope.

28. The nanoparticle of claim 27, wherein the cancer epitope is a cancer neoantigen epitope.

29. The nanoparticle of claim 27 or 28, wherein the cancer neoantigen epitope comprises a cancer epitope selected from among ME-1. M26. Gludl. Mtchl. Ml 8. and E2f8.

30. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecules comprises a sequence-specific nuclease.

31. The nanoparticle of claim 30, wherein the sequence-specific nuclease is a Cas nuclease.

32. The nanoparticle of claim 31, wherein the Cas nuclease is a Cas9 nuclease, a CasX nuclease, a Casl2 nuclease, or a Casl3 nuclease.

33. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecule comprises an antibody or an antigen-binding fragment thereof.

34. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecule comprises an immunomodulatory polypeptide.

35. The nanoparticle of claim 34, wherein the immunomodulatory polypeptide comprises a cytokine.

36. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecules comprises a transcription factor.

37. The nanoparticle of claim 23 or 24, wherein the one or more exogenous molecules comprises a reporter molecule.

38. The nanoparticle of claim 37, wherein the reporter molecule comprises a firefly luciferase, an enhanced green fluorescent protein (eGFP) or a red fluorescent protein (RFP).

39. The nanoparticle of any of claims 1-38, wherein when the nanoparticle is administered to a subject, an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle.

40. The nanoparticle of claim 39, wherein the immune response comprises an increase in cytokine production.

41. The nanoparticle of claim 40, wherein the cytokine is selected from among one or more of: IFN-y, IL-2, IL-6, and IL- 12.

42. The nanoparticle of claim 39, wherein the immune response comprises an increase in co-stimulatory molecule expression on dendritic cells.

43. The nanoparticle of claim 42, wherein the co-stimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.

44. The nanoparticle of claim 39, wherein the immune response comprises activation of immune cells.

45. The nanoparticle of claim 44, wherein the immune cell is selected from among one or more of: NKT, NK. CD4+T, and CD8+T cells.

46. The nanoparticle of any of claims 39-45, wherein the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity of tumor-specific T cells, and increase in migration of tumor-specific T cells.

47. The nanoparticle of any of claims 39-46, wherein the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.

48. The nanoparticle of any of claims 1-47, wherein when administered to a subject, the nanoparticle is localized to the spleen.

49. The nanoparticle of any of claims 1-48, wherein when administered to a subject, the nanoparticle is localized to a tumor.

50. The nanoparticle of any of claims 1-49, wherein when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node.

51. The nanoparticle of any of claims 1-50, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen.

52. The nanoparticle of any of claims 1-51, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor.

53. The nanoparticle of any of claims 1-52, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor-draining lymph node.

54. The nanoparticle of any of claims 1-53, wherein when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor growth, or increasing survival of the subject.

55. A method for generating a nanoparticle, the method comprising generating a lipid mixture of N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE); and incorporating an a galactosylceramide to the mixture.

56. The method of claim 55, wherein the galactosylceramide is alphagalactosylceramide (a-GalCer).

57. The method of claim 55 or 56, wherein The method forms a lipoplex (LPX).

58. The method of any of claims 55-57, wherein the ratio of DOTMA:DOPE is between about 5: 1 and about 1 :5.

59. The method of any of claims 55-58, wherein the ratio of DOTMA:DOPE is between about 3: 1 and about 1 :3.

60. The method of any of claims 55-59, wherein the ratio of DOTMA:DOPE is about2: 1.

61. The method of any of claims 55-60, wherein the proportion of a-GalCer is between about 0.1% and about 5% molar percentage.

62. The method of any of claims 55-61, wherein the proportion of a-GalCer is between about 0.5% and about 2% molar percentage.

63. The method of any of claims 55-62, wherein the proportion of a-GalCer is about 0.5% molar percentage.

64. The method of any of claims 55-62, wherein the proportion of a-GalCer is about 1% molar percentage.

65. The method of any of claims 55-62, wherein the proportion of a-GalCer is about 2% molar percentage.

66. The method of any of claims 55-65, further comprising mixing the nanoparticle with a nucleic acid.

67. The method of claim 66, wherein the nucleic acid comprises a ribonucleic acid (RNA).

68. The method of claim 65 or 66, wherein the nucleic acid comprises a circular RNA.

69. The method of claim 65 or 66, wherein the nucleic acid comprises a linear RNA.

70. The method of any of claims 65, 66, and 69. wherein the nucleic acid comprises a cleaved linear RNA comprising a hydroxyl group at the 5' terminus, and a 2',3'-cyclic phosphate at the 3' terminus.

71. The method of any of claims 65, 66, and 69. wherein the nucleic acid comprises a linear messenger RNA (mRNA).

72. The method of any of claims 65, 66, and 69, wherein the nucleic acid comprises a micro RNA (miRNA).

73. The method of any of claims 65, 66, and 69. wherein the nucleic acid comprises a small inhibitory RNA (siRNA).

74. The method of any of claims 65, 66, and 69, wherein the nucleic acid comprises a guide RNA (gRNA).

75. The method of any of claims 66-74, wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is between about 1:2 to about 2: 1.

76. The method of any of claims 66-75, wherein the nucleic acid is a ribonucleic acid (RNA), and the DOTMA:RNA charge ratio is about 1.3:2.

77. A nanoparticle generated by the method of any of claims 55-76.

78. A composition comprising the nanoparticle of any of claims 1-54 and 77.

79. The composition of claim 78, wherein the composition is a pharmaceutical composition.

80. The composition of claim 78 or 79, wherein the composition comprises a pharmaceutically acceptable excipient.

81. A method of vaccinating a subject, the method comprising administering the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 to a subject.

82. A method for enhancing an immune response to a vaccine epitope in a subject, the method comprising administering the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 to a subject.

83. A method of treating a disease or disorder in a subject, the method comprising administering the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 to a subject.

84. The method of any of claims 81-83, wherein an immune response to the vaccine epitope is increased compared to a subject without administration of the nanoparticle or the composition.

85. The method of claim 84, wherein the immune response comprises an increase in cytokine production.

86. The method of claim 85, wherein the cytokine comprises one or more of: IFN-y, IL- 2, IL-6, and IL-12.

87. The method of claim 85 or 86, wherein the cytokine comprises IFN-y.

88. The method of any of claims 84-87, wherein the immune response comprises an increase in co-stimulatory molecule expression on dendritic cells.

89. The method of claim 88, wherein the immune response comprises an increase in costimulatory molecule expression on MHC-l CDl Ib’CDl lc+dendritic cells.

90. The method of claim 88, wherein the immune response comprises an increase in costimulatory molecule expression on MHC-II+CD1 lb+CDl lc+dendritic cells.

91. The method of any of claims 88-90, wherein the co-stimulatory molecule is selected from among one or more of: CD40, CD80, and CD86.

92. The method of any of claims 84-91, wherein the immune response comprises activation of an immune cell.

93. The method of claim 92, wherein the immune cell is selected from among one or more of: NKT, NK, CD4+T. and CD8+T cells.

94. The method of any of claims 84-91, wherein the immune response comprises one or more of: the generation and proliferation of tumor-specific T cells, increase in cytolytic activity7of tumor-specific T cells, and increase in migration of tumor-specific T cells.

95. The method of any of claims 84-94, wherein the immune response comprises developing an immune tolerance to an epitope involved in an autoimmune disease or disorder.

96. The method of any of claims 81-95, wherein when administered to a subject, the nanoparticle is localized to the spleen.

97. The method of any of claims 81-96, wherein when administered to a subject, the nanoparticle is localized to a tumor.

98. The method of any of claims 81-97, wherein when administered to a subject, the nanoparticle is localized to a tumor-draining lymph node.

99. The method of any of claims 81-98, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to the spleen.

100. The method of any of claims 81-99, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor.

101. The method of any of claims 81-100, wherein when administered to a subject, the nanoparticle is capable of delivering the nucleic acid to a tumor-draining lymph node.

102. The method of any of claims 81-101, wherein when administered to a subject, the nanoparticle is capable of reducing tumor volume, inhibiting tumor growth, or increasing survival of the subject.

103. The method of any of claims 81-102, wherein the method prevents or reduces the severity of a disease or disorder associated with the vaccine epitope in the subject.

104. The method of claim 103, wherein the disease or disorder is an infectious disease, optionally a viral infection.

105. The method of claim 103 or 104, wherein the disease or disorder is a cancer.

106. The method of any of claims 81-105, wherein the administration is by intravenous administration.

107. The nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 for use in vaccinating a subject, wherein the nanoparticle, or the composition is administered to the subject.

108. The nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 for use in enhancing an immune response to a vaccine epitope in a subject, wherein the nanoparticle, or the composition is administered to the subject.

109. The nanoparticle of any of claims 1-54 and 77. or the composition of any of claims 78-80 for use in treating a disease or disorder in a subject wherein the nanoparticle, or the composition is administered to the subject.

110. Use of the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 in the manufacture of a medicament for vaccinating a subject, wherein the medicament is administered to the subject.

111. Use of the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 in the manufacture of a medicament for enhancing an immune response to a vaccine epitope in a subject, wherein the medicament is administered to the subject.

112. Use of the nanoparticle of any of claims 1-54 and 77, or the composition of any of claims 78-80 in the manufacture of a medicament for treating a disease or disorder in a subject, wherein the medicament is administered to the subject.

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