Lipid nanoparticles and methods of use

Abstract

The present disclosure relates to lipids and compositions thereof. In various aspects of the disclosure, the compositions are lipid nanoparticle compositions that are used as adjuvants used with vaccines, such as RNA vaccines, and / or to prevent or treat diseases or disorders in a subject in need thereof.

Description

LIPID NANOPARTICLES AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 707,747, which was filed October 15, 2024, is titled Lipid Nanoparticles and Methods of Use, and is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This disclosure was made with government support under grant numbers 75N93019C00051, AI153064, AI165066, and AI166916 awarded by the National Institutes of Health. The government has certain rights in this disclosure. STATEMENT SEQUENCE LISTING

[0003] The sequence filed herewith, titled WIST-015-PCT_SL.xml, created on October 2, 2025, and having a file size of 19,559 bytes is incorporated herein in its entirety. BACKGROUND

[0004] Vaccination is critical for disease control and prevention. Disease correlates of protection are frequently unknown, lending value to vaccine approaches that can elicit both arms of adaptive immunity. Gene-vectored vaccines, such as nucleic acid and viral-vectored, have the capacity to elicit both humoral and cellular immunity (1-4). Vaccine approaches that can elicit both arms of adaptive immunity are valuable for their potential promise against diverse pathogens as correlates of protection are frequently complex or unvalidated. Genetic vaccines, such as nucleic acid and viral-vectored, can elicit both humoral and cellular immunity (1-4) and have production advantages.

[0005] Lipid nanoparticle-formulated nucleoside-modified mRNA vaccines (mRNA- LNPs) have demonstrated efficacy for the prophylaxis of SARS-CoV-2, with two initial product licenses from Moderna (mRNA-1273 / SpikeVax) and Pfizer-BioNTech(BNT162b2 / Comirnaty) (5, 6). Immunization with mRNA-LNPs in animal models and humans is associated with robust immunogenicity (3, 7). Translation of mRNA has been demonstrated to be through a strong, short burst of antigen production (8). The introduction of modified nucleosides has been shown to reduce innate reactogenicity and improve translation efficiency of in vitro transcribed (IVT) RNA (8, 9). Recently, the activity of the LNP component to both adjuvant as well as to deliver nucleoside-modified mRNA vaccines has been described (10-12).

[0006] IVT mRNA reactions have distinct synthetic requirements including template DNA, RNA polymerase, modified and / or unmodified ribonucleotides to form mRNA transcripts as well as a process to add a 5’ cap structure (13, 14). After transcription, IVT mRNA requires purification to remove byproducts of the reaction, such as double-stranded RNA species (15).

[0007] Additionally, mRNA thermostability leads to cold chain dependent storage requirements. While there has been approaches to improve mRNA-LNP vaccine stability such as lyophilization (16), additional vaccine approaches with reduced synthetic requirements could be valuable.

[0008] DNA vaccines have demonstrated clinical safety and efficacy while also conferring advantages relative to some platforms including temperature stability combined with simple production (17-22). Naked DNA is poorly immunogenic, and advancements in device delivery have improved in vivo immunogenicity. Physical transfection modalities for DNA vaccines include in vivo electroporation (EP), gene gun, and jet delivery among others; a licensed DNA vaccine for SARS-CoV-2 (ZyCoV-D) utilizes jet delivery (23-25). The direct encoding of gene sequences (e.g., cytokines) to further adjuvant DNA has shown further improvement in immunogenicity through induction of enhanced humoral and cellular responses in the clinic (26-28). Additional methods of further improving in vivo DNA vaccine immunogenicity would be important, considering the positive product profile of DNA.

[0009] Lipid nanoparticle formulations of DNA for in vivo delivery have been studied, and formulations result in robust transfection efficiency (29-31). However, translating lipid- mediated transfection of DNA in vivo to immunogenicity in the clinic has been challenging(32). It has been hypothesized that DNA delivered in the cytoplasm is inefficiently transferred to the nucleus; early studies using naked plasmid DNA as vaccines for delivery suggest that this model should be revisited (33, 34). Similarly, ionizable LNPs, including those formulated with SM-102 (Moderna formulation) and ALC-0315 (Pfizer- BioNTech formulation) have had limited success at inducing immune responses when formulated with plasmid DNA encoding firefly luciferase (35). SUMMARY OF EMBODIMENTS

[0010] In some embodiments, the disclosure relates to a method of enhancing an immune response in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition comprising a first lipid nanoparticle (LNP) and a nucleic acid molecule comprising DNA. In some embodiments, the LNP comprises SM-102 or a derivative thereof. In some embodiments, the LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the LNP comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the LNP comprises polyethylene glycol (PEG). In some embodiments, the LNP comprises (i) SM-102 or a derivative thereof, (ii) a cholesterol molecule or a derivative thereof, (iii) 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or a derivative thereof, and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, (ii), (iii), (iv) and (v) form a lipid nanoparticle encapsulating the nucleic acid molecule. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof. In some embodiments, the method further comprises administering a second lipid nanoparticle comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or a functional variant thereof. In some embodiments, the antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease. In some embodiments, the composition further comprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.1 micrograms to about 400 micrograms. In some embodiments, thecomposition further comprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms. In some embodiments, the lipid to DNA weight ratio of the first lipid nanoparticle is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1. In some embodiments, the nucleic acid molecule comprising DNA comprises one or a plurality of: a cDNA molecule, a linear DNA molecule, a circular plasmid DNA molecule, mini-circle DNA molecule, a rolling circle amplified DNA product, an artificial chromosome, a replicating DNA or any combination thereof. In some embodiments, the first lipid nanoparticle comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension.

[0011] In some embodiments, the first lipid nanoparticle comprises a spheroid shape and comprises a diameter from about 50 to about 180 nanometers at its longest width dimension. In some embodiments, the method comprises a population of first lipid nanoparticles encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules. In some embodiments, the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 3 nucleic acid molecules. In some embodiments, the nucleic acid molecule comprising DNA is a DNA plasmid. In some embodiments, the first lipid nanoparticle has a zeta potential of from about - 2 millivolts to about -22 millivolts. In some embodiments, the composition comprises a plurality of first lipid nanoparticles; and wherein the first lipid nanoparticles have an average zeta potential of from about -0.5 mV to about -6 mV. In some embodiments, the composition comprises a plurality of first lipid nanoparticles. In some embodiments, the first lipid nanoparticles have an average diameter of from about 70 to about 77 nanometers.

[0012] In some embodiments, the nucleic acid molecule comprising DNA is free of RNA. In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding one or a plurality of antigenic determinants or functional variants thereof.

[0013] In some embodiments, the immune response is against an antigenic determinant or functional variant thereof. In some embodiment, enhancing an immune response comprisesenhancing a immune response to a vaccine. In some embodiment, enhancing an immune response comprises enhancing an antigen-specific immune response in a subject in need thereof before, after or simultaneous administration of a vaccine comprising an antigenic determinant or functional variant thereof or a nucleic acid sequence encoding an antigenic determinant or functional variant thereof.

[0014] The disclosure also relates to a method of vaccinating a subject in need thereof. In some embodiments, the method comprises administering a composition to the subject, wherein the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof and at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, the method comprises administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises an RNA molecule encoding an antigenic determinant or functional variant thereof. In some embodiments, the second composition comprises at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNPencapsulates a nucleic acid molecule comprising DNA. In some embodiments, the at least first LNP comprises (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the at least first LNP comprises nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the at least first LNP comprises PEG is DMG-PEG-2000 or a derivative thereof. In some embodiments, the at least first LNP comprises method further comprises a second lipid nanoparticle encapsulating the RNA molecule. In some embodiments, the at least first LNP comprises antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease. In some embodiments, the at least first LNP comprises composition further comprises a plurality of LNPS comprising DNA with a total mass amount of from about 0.1 micrograms to about 400 micrograms of DNA In some embodiments, the at least first LNP comprises composition further comprises one or a plurality of LNPs comprising nucleic acid molecules comprising DNA with a total mass amount of from about 0.002 micrograms to about 4 micrograms of DNA. In some embodiments, the at least first LNP comprises lipid to DNA weight ratio of the LNP is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1. In some embodiments, the at least first LNP comprises nucleic acid molecule comprising DNA is a DNA plasmid. In some embodiments, the at least first LNP comprises at least first LNP comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension. In some embodiments, the at least first LNP comprises composition comprises a population of first LNPs encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first LNPs are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules. In some embodiments, the at least first LNP comprises LNP comprising a nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a variant thereof. In some embodiments, the at least first LNP comprises LNP comprising a nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding any antigenic determinant or a variant thereof.

[0015] In some embodiments, the disclosure relates to a composition comprising a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encodingan antigenic determinant or functional variant thereof; and a second LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, the second LNP comprises SM-102 or a derivative thereof. In some embodiments, the second LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the second LNP comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the second LNP comprises polyethylene glycol (PEG). In some embodiments, the second LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of any nucleic acid sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, the first LNP comprises SM-102 or a derivative thereof. In some embodiments, the first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first LNP comprises polyethylene glycol (PEG). In some embodiments, the first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG- PEG-2000 or a derivative thereof.

[0016] In some embodiments, the disclosure relates to a pharmaceutical composition comprising (a) an effective amount of a composition and (b) a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; and a second LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, the second LNP comprises SM-102 or a derivative thereof. Insome embodiments, the second LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the second LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the second LNP comprises polyethylene glycol (PEG). In some embodiments, the second LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of any nucleic acid sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, the first LNP comprises SM-102 or a derivative thereof. In some embodiments, the first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first LNP comprises polyethylene glycol (PEG). In some embodiments, the first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof.

[0017] In some embodiments, the disclosure relates to a pharmaceutical composition comprising: (a) a first LNP comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; (b) a second LNP comprising a DNA molecule comprising a nucleic acid sequence free of any antigenic determinant or functional variant thereof; and (c) a pharmaceutically acceptable carrier. In some embodiments, the first and second LNPs comprise SM-102 or a derivative thereof. In some embodiments, the first and second LNPs comprise a cholesterol molecule or a derivative thereof. In some embodiments, the first and second LNPs comprise 1,2-distearoyl-sn-glycero-3-phosphocholine(DSPC) or a derivative thereof. In some embodiments, the first and second LNPs comprise polyethylene glycol (PEG). In some embodiments, the first and second LNPs comprise: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the RNA sequence encodes a viral antigenic determinant or an antigenic determinant associated with hyperproliferative disease.

[0018] In some embodiments, the disclosure relates to a method of vaccinating a subject in need thereof . In some embodiments, the method comprises (a) administering to the subject a composition comprising a vaccine and a vaccine adjuvant; or (b) administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises a vaccine and the second composition comprises a vaccine adjuvant.

[0019] In some embodiments, the vaccine comprises an antigenic determinant or a functional variant thereof or comprises a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the vaccine adjuvant comprises at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, the vaccine comprises an RNA molecule comprising an RNA sequence that encodes the antigenic determinant or functional variant thereof. In some embodiments, the vaccine comprises a lipid nanoparticle comprising from about 1 to about 20 RNA molecules. In some embodiments, the nucleic acid molecule comprising DNA is free of RNA. In someembodiments, the nucleic acid molecule comprising DNA is free of a DNA sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

[0020] In some embodiments, the disclosure relates to a method of preventing or treating a pathogen infection in a subject in need thereof. In some embodiments, the method comprises (a) administering a composition to the subject. In some embodiments, the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof from the pathogen and at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA.

[0021] In some embodiments, the method of preventing or treating a pathogen infection comprises (b) administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises an RNA molecule encoding an antigenic determinant or functional variant thereof. In some embodiments, the second composition comprises at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i),(ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof.

[0022] In some embodiments, the method of preventing or treating a pathogen infection further comprises a second lipid nanoparticle encapsulating the RNA molecule. In some embodiments, the method of preventing or treating a pathogen infection, the DNA molecule is free of a nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

[0023] In some embodiments, the disclosure relates to a method of enhancing an antigen-specific immune response in a subject in need thereof. In some embodiments, the method comprises (a) administering a vaccine comprising an antigenic determinant or functional variant thereof or administering a vaccine comprising a nucleic acid sequence encoding the antigenic determinant or functional variant thereof; and (b) administering an LNP comprising a DNA molecule is free of a nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the LNP comprises SM-102 or a derivative thereof. In some embodiments, the LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the LNP comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the LNP comprises polyethylene glycol (PEG). In some embodiments, the LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG In some embodiments, (i), (ii), (iii) and (iv) form the LNP and the LNP encapsulates the DNA molecule. In some embodiments, the DNA molecule is free of a nucleic acid sequence encoding any antigenic determinant or functional variant thereof.

[0024] In some embodiments, the disclosure relates to a method of treating or preventing an infection or a disorder in a subject in need thereof. In some embodiments, the method comprising administering to the subject a pharmaceutical composition herein. In some embodiments, if the method treats or prevents an infection, the antigenic determinant orfunctional variant thereof is from a pathogen. In some embodiments, if the method treats or prevents a disorder, the antigenic determinant or functional variant thereof is associated with the disorder. In some embodiments, the disorder is a hyperproliferative disorder and the antigenic determinant or functional variant thereof is from a cell associated with a hyperproliferative disorder.

[0025] In some embodiments, the disclosure relates to a cell comprising any composition(s) or any pharmaceutical composition(s) herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following detailed description of preferred embodiments of the disclosure will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the disclosure, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the disclosure is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0027] FIG. 1A–FIG. 1C depict the ratio of lipid to DNA formulation impacts immunogenicity. FIG.1A depicts representative FACS plots of Tfh cells from mice immunized with 2 µg of pVAX DNA-LNP or CA09 HA DNA-LNP formulated at a 40:1, 20:1, or 10:1 Lipid to DNA ratio. Bar plots show quantification of frequency of Tfh cells at 14 days post immunization in the draining lymph nodes. Frequency is expressed as a percentage of activated (CD44+) CD4+ T cells. Pre-gated on live CD19- CD4+. FIG.1B depicts data demonstrating the total GC B cells as in (FIG.1A). Frequency expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ (FIG. 1C) CA09 HA-specific GC B cells as in (FIG. 1A). Frequency expressed as a percentage of CD38- Fas+ GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+. Dots represent individual animals; n=4-8 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0028] FIG. 2A–FIG. 2C depict data demonstrating that the influenza virus HA- expressing DNA-LNP induces robust GC responses. FIG. 2A depicts representative FACSplots of Tfh cells from mice immunized with 2 µg of pVAX DNA-LNP, CA09 HA DNA-LNP, or CA09 HA mRNA-LNP. Bar plots show quantification of frequency (left) and numbers (right) of Tfh cells at 14 days post immunization in the draining lymph nodes. Frequency is expressed as a percentage of activated (CD44+) CD4+ T cells. Pre-gated on live CD19- CD4+. FIG.2B depicts the total GC B cells as in (FIG.2A). Frequency expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ (FIG.2C) CA09 HA-specific GC B cells as in (FIG.2A). Frequency expressed as a percentage of CD38- Fas+ GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+. Dots represent individual animals; n=10 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons withBonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0029] FIG. 3A–FIG. 3H depict data demonstrating that Influenza HA DNA-LNP induces serum responses comparable to mRNA-LNP and adjuvanted protein and is thermostable. FIG.3A depicts the area under the curve (AUC) of biweekly serum ELISA data. FIG. 3B depicts the serum endpoint titers at week 8 to A / California / 04 / 2009 HA. FIG. 3C depicts the HAI titers at week 8 to A / California / 07 / 2009 X-179A. FIG. 3D–FIG. 3H depict data demonstrating serum endpoint titers at week 8 to A / Michigan / 45 / 2015 HA (FIG. 3D), A / Wisconsin / 588 / 2019 HA (FIG. 3E), A / Sydney / 5 / 2021 HA (FIG. 3F), and A / Victoria / 4897 / 2022 HA (FIG.3G). FIG. 3H depicts the serum endpoint titers at week 4 to A / California / 04 / 2009 HA from mice immunized with DNA-LNP preparations under indicated storage conditions. Dots represent individual animals; for a-g, n=10 animals per group; for h, n=5-15 animals per group. For FIG.3B–FIG.3H depict plots showing geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare active immunization groups. * p<0.05, ** p<0.01, *** p<0.001,**** p<0.0001.

[0030] FIG.4A–FIG.4I depict data demonstrating that Influenza HA DNA-LNP elicits potent antigen-specific CD8+ and CD4+ T cell responses. Representative FACS plots of IFNv+ CD8+ T cells. Bar plot shows quantification of frequency expressed as a percentage of activated (CD44+ CD62L-) CD8+ T cells in the spleen 14 days post immunization. Pregatedon live CD3+ CD4- CD8+ CD44+ CD62L-. (FIG. 4B) CD107a+ CD8+ T cells shown as in (FIG.4A). (FIG.4C) TNFα+ CD8+ T cells shown as in (FIG.4A). (FIG.4D) IFNv+ CD4+ T cells expressed as a percentage of activated (CD44+ CD62L-) CD4+ T cells in the spleen 14 days post immunization. Pregated on live CD3+ CD8- CD4+ CD44+ CD62L-. (FIG. 4E) TNFα+ CD4+ T cells shown as in (FIG.4D). (FIG.4F) IL-2+ CD4+ T cells shown as in (FIG. 4D). FIG. 4G - FIG. 4I depict data demonstrating the frequency of activated CD8+ T cells expressing IFNγ (FIG. 4G), CD107a (FIG. 4H), or TNFα (FIG. 4I) after immunization with varying doses of HA DNA-LNP or mRNA-LNP as indicated. Pregated on live CD3+ CD4- CD8+ CD44+ CD62L-. Dots represent individual animals; for FIG.4A - FIG.4F, n=10 animals per group; for FIG.4G - FIG.4I, n=5 animals per group. For FIG. 4A - FIG.4F, plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0031] FIG. 5A–FIG. 5H depict data demonstrating that SARS-CoV-2 spike- expressing DNA-LNP elicits robust GC and serum antibody responses. (FIG. 5A) Representative FACS plots of Tfh cells from mice immunized with 2 µg of pVAX DNA-LNP, spike DNA-LNP, spike mRNA-LNP, or BNT162b2. Bar plots show quantification of frequency of Tfh cells at 14 days post immunization in the draining lymph nodes. Frequency is expressed as a percentage of activated (CD44+) CD4+ T cells. Pre-gated on live CD19- CD4+. (FIG.5B) Total GC B cells as in (FIG.5A). Frequency expressed as a percentage of CD19+ B cells. Pre- gated on live CD4- CD19+ (FIG. 5C) Spike-specific GC B cells as in (FIG.5A). Frequency expressed as a percentage of CD38- Fas+ GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+ (FIG. 5D- FIG. 5H) Serum endpoint titers at Week 4 post immunization to wild-type spike RBD (FIG.5D), D614G full length spike (FIG.5E), B.1.617.2 (Delta) full length spike (FIG. 5F), and BA.2 full length spike (FIG. 5G). (FIG. 5H) Serum neutralization ID50 against wild-type SARS-CoV-2 pseudovirus. Dots represent individual animals; n=4-8 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used tocompare groups (FIG. 5A - FIG.5C) or active immunization groups (FIG.5D - FIG. 5H). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0032] FIG. 6A–FIG. 6F depict data demonstrating that Spike DNA-LNP induces potent T cell responses. FIG. 6A depicts representative FACS plots of IFNv+ CD8+ T cells. Bar plot shows quantification of frequency expressed as a percentage of activated (CD44+ CD62L-) CD8+ T cells in the spleen 14 days post immunization. Pregated on live CD3+ CD4- CD8+ CD44+ CD62L-. (FIG.6B) CD107a+ CD8+ T cells shown as in (FIG.6A). (FIG.6C) TNFα+ CD8+ T cells shown as in (FIG.6A). (FIG.6D) Representative FACS plots of IFNv+ CD4+ T cells. Bar plot shows quantification of frequency expressed as a percentage of activated (CD44+ CD62L-) CD4+ T cells in the spleen 14 days post immunization. Pregated on live CD3+ CD8- CD4+ CD44+ CD62L-. (FIG.6E) TNFα+ CD4+ T cells shown as in (FIG. 6D). (FIG.6F) IL-2+ CD4+ T cells shown as in (FIG.6D). Dots represent individual animals; n=4-8 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0033] FIG.7A and FIG.7B depict data demonstrating that mouse muscle cells can be effectively transfected with lipid-formulated plasmid DNA. (FIG.7A–FIG.7B) C2C12 cells (mouse myoblast) were transfected via lipofectamine with plasmid DNA expressing GFP (FIG. 7A) or empty vector pVAX (FIG.7B). Representative images of cells under light microscopy or fluorescence (470 nm excitation, 525 nm emission). Experiment repeated at least twice.

[0034] FIG.8A and FIG.8B depict data demonstrating that the longevity of antibody titers in mice immunized with HA DNA-LNP. (FIG.8A–FIG.8B) Mice were immunized with 2µg of HA DNA-LNP and serum titers were followed longitudinally, reported as raw OD (FIG. 8A) or endpoint titer calculated against naïve animals (FIG. 8B). Dots represent individual animals; n=15 animals per group. Plots show geometric mean with geometric SD.

[0035] FIG. 9A–FIG. 9C depict data demonstrating the dosing of DNA-LNP and mRNA-LNP elicits similar CD4+ T cell responses. (FIG.9A–FIG.9C) Frequency of activated CD4+ T cells expressing IFNv (FIG. 9A), TNFα (FIG. 9B), or IL-2 (FIG. 9C) after immunization with varying doses of HA DNA-LNP or mRNA-LNP as indicated. Pregated onlive CD3+ CD8- CD4+ CD44+ CD62L-. Dots represent individual animals; n=5 animals per group. Plots show geometric mean with geometric SD.

[0036] FIG.10A–FIG.10C depict data demonstrating the absolute numbers of Tfh and GC B cells after immunization with Spike-expressing DNA or mRNA-LNPs. (FIG.10A–FIG. 10C) Mice were immunized as in FIG.1. Bar plots show quantification of absolute numbers of Tfh (FIG.10A), total GC B (FIG.10B), and spike-specific GC B (FIG.10B) in DLNs 14 days post immunization. Dots represent individual animals; n=4-8 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05. Related to FIGS.1A–1C.

[0037] FIGS. 11A–11I illustrate initial immune characterization of HA DNA-LNP formulations and immunogenicity. FIG.11A: Representative FACS plots of GC B cells from mice immunized with 2 μg of pVAX DNA-LNP or CA09 HA DNA-LNP formulated at a 10.5, 5.3, or 2.6 N / P ratio. FIG.11B: Bar plots quantifying frequency of GC B cells at 14 days post immunization in the DLNs. Frequency expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ FIG. 11C: Frequency of CA09 HA-specific GC B cells expressed as a percentage of CD38- Fas+ GC B cells. Pre-gated on live CD4- CD19+ CD38- Fas+. FIG.11D: Frequency of activated Tfh cells expressed as a percentage of CD44+ CD4+ T cells. Pre-gated on live CD19- CD4+. FIG. 11E: IFNγ ELISpot of splenocytes 14 days post immunization. FIGS.11F and 11G: Fold change induction of cytokines in DLNs at 4 hours (FIG.11F) and 24 hours (FIG.11G) post immunization quantified using Luminex. FIGS.11H and 11I: ELISpot assay measuring IFNα (FIG. 11H) and IFNγ (FIG. 11I) 20 hours post stimulation of splenocytes ex vivo with DNA-LNP, plasmid DNA, or DNA-LNP in the presence of chemical inhibitors to the indicated DNA sensors. FIG.11J: Schematic of relevant pathways implicated in driving inflammation from DNA-LNPs. Dots represent individual animals; n=8-9 (a-e), n=5 (f-g), or n=3-4 animals per group (FIGS. 11H and 11I); data pooled from two independent experiments (FIGS.11A–11E, 11H, and 11I) or from one independent experiment (FIGS.11F and 11G). Plots show geometric mean with geometric SD. Unpaired one-way ANOVAadjusted for multiple comparisons with Bonferroni corrections was used to compare groups or compared to DNA-LNP control (h-i) * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0038] FIGS. 12A–12M illustrate Early immune profiling of innate and adaptive populations reveals robust activation status after HA DNA-LNP immunization. FIG. 12A: Representative FACS plots of CD69+ CD8+ T cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3+ NKp46-. FIG.12B: Bar plot showing quantification of CD69+ CD8+ T cells as in (a) in the popliteal DLN, iliac DLN, and spleen. FIG. 12C: Representative FACS plots of CD69+ NK cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3- NKp46+. FIG.12D: Bar plot showing quantification of CD69+ NK cells as in (FIG.12C) in the popliteal DLN, iliac DLN, and spleen. FIG.12E: Schematic of immunization and analysis scheme. Mice were immunized once with 2μg HA DNA-LNP, HA mRNALNP, or 1μg HA protein in Addavax and sacrificed 24 hours later to examine innate immune populations. Cells were analyzed by flow cytometry (DLN and spleen) or single cell transcriptomics (DLN only). FIGS. 12F–12K: Frequency of total mDCs (f), CD11b+ mDCs (FIG.12G), CD103+ mDCs (h), pDCs (FIG.12I), neutrophils (FIG.12J), and monocytes (FIG. 12K) in the iliac DLN expressed as a frequency of CD45+ cells. FIG. 12L: Representative histograms of CD86 expression on total mDC and mDC subpopulations after immunization with the indicated immunogens. FIG.12M: Quantification of CD86 expression on all innate populations in FIGS.12F–12K. Dots represent individual animals; for (ad), n=4-5 animals per group; for (FIGS. 12F–12M), n=9-10 animals per group. Data representative of one (FIGS. 12A–D) or two (FIGS.12F–12M) independent experiment(s). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups in (b, d, f-k, m). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0039] FIGS.13A–13G illustrated single cell transcriptomics elucidates pro-activation and migration signature among innate immune subsets after priming with HA DNA-LNP. FIG. 13A: UMAP plot of innate immune subsets. FIG. 13B: UMAP plot of clusters colored by sample. FIGS.13C–13G: Differentially expressed genes upregulated after immunization with DNA-LNP relative to naïve, mRNA-LNP, and protein in adjuvant immunization in NK cells(FIG.13C), neutrophils (FIG.13D), monocytes (FIG.13E), pDCs (FIG.13F), and cDCs (FIG. 13G). Data represent one independent experiment of 10 pooled mouse popliteal LNs per group.

[0040] FIGS.14A–14J illustrate Influenza virus HA DNA-LNP elicits potent antigen- specific CD8+ and CD4+ T cell responses. FIG. 14A: Representative FACS plots of IFNγ+ CD8+ effector T cells. FIG. 14B: Quantification of frequency expressed as a percentage of activated (CD44+ CD62L-) CD8+ T cells in the spleen 14 days post immunization. Pregated on live CD3+ CD4- CD8+ CD44+ CD62L-. FIG.14C: CD107a+ CD8+ effector T cells. FIG. 14D: TNFα+ CD8+ effector T cells. FIG.14E: IFNγ+ CD4+ T cells expressed as a percentage of activated (CD44+ CD62L-) CD4+ T cells in the spleen 14 days post immunization. Pregated on live CD3+ CD8- CD4+ CD44+ CD62L-. FIG. 14F: TNFα+ CD4+ effector T cells. FIG. 14G: IL-2+ CD4+ effector T cells. FIGS. 14H–14J: Frequency of activated CD8+ T cells expressing IFNγ (h), CD107a (i), or TNFα (j) after immunization with varying doses of HA DNA-LNP or mRNA-LNP as indicated. Pregated on live CD3+ CD4- CD8+ CD44+ CD62L- . Dots represent individual animals; for (FIGS.14A–14G), n=10 animals per group; for (FIGS. 14H–14J), n=5 animals per group. Data pooled from two (FIGS. 14A–14G) or one (FIGS. 14H–14J) independent experiment(s). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0041] FIGS.15A–15P illustrates Influenza virus HA-expressing DNA-LNP induces robust GC and serum responses. FIG.15A: Representative FACS plots of Tfh cells from mice immunized with 2 μg of pVAX DNA-LNP, CA09 HA DNA-LNP, or CA09 HA mRNA-LNP. (b-c) Bar plots show quantification of frequency (FIG. 15B) and numbers (FIG.15C) of Tfh cells at 14 days post immunization in the draining lymph nodes. Frequency is expressed as a percentage of activated (CD44 +) CD4+ T cells. Pre-gated on live CD19- CD4+. FIG.15D: Representative FACS plots of total GC B cells. (e-f) Bar plots show quantification of frequency (FIG.15E) and numbers (FIG.15F). Frequency is expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ (FIG.15G) Representative FACS plots of CA09 HA-specific GC B cells. FIGS.15H and 15I: Bar plots show frequency (FIG.15H) and numbers (FIG.15I). Frequency expressed as a percentage of CD38- Fas+ GC B cells. Pre-gated on live CD4-CD19+ CD38- Fas+. FIG.15J: Area under the curve (AUC) of total A / California / 04 / 2009 HA- specific serum IgG ELISA data. DPI, Days post immunization. FIG. 15K: Serum endpoint titers at week 8 to various H1N1 HAs. FIG.15L: HAI titers at week 8 to A / California / 07 / 2009 X-179A. (m-n) AUC of serum binding antibodies to A / Guangdong-Maonan / SWL1536 / 2019 (FIG. 15M), and A / Victoria / 4897 / 2022 (FIG. 15N). FIGS. 15O and 15P: HAI titers to A / Netherlands / 602 / 2009 (FIG. 15O) and A / New York City / PV63249 / 2022 (FIG. 15P). Dots represent individual animals; n=9-10 animals per group; data pooled from two independent experiments. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups (a- i) or active immunization groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0042] FIGS. 16A–16I illustrate SARS-CoV-2 spike-expressing DNA-LNP elicits serum antibody responses and is protective in lethal challenge. FIG. 16A: Schematic of immunization and challenge scheme. Mice were immunized once with 2 μg of empty vector pVAX DNA-LNP, spike DNA-LNP, or spike mRNA-LNP. Serology was performed 8 weeks post prime, and animals were challenged approximately 9 weeks post prime. FIGS.16B–16E: Serum endpoint titers at Week 8 post immunization to wild-type spike RBD (FIG. 16B), D614G full length spike (FIG.16C), B.1.617.2 (Delta) full length spike (FIG.16D), and BA.2 full length spike (FIG. 16E). FIG. 16F: Serum neutralization ID50 against wild-type SARS- CoV-2 pseudovirus. FIGS.16G and 16H: Weight loss (FIG.16G) and survival (FIG.16H) of mice challenged with 1x105 PFU mouse-adapted SARS-CoV-2 with an 80% weight loss cutoff. (FIG.16I) Clinical score representing clinical signs of morbidity 4 days post challenge. Dots represent individual animals; n=5-8 animals per group; data combined from of two independent studies (a-f). Plots show geometric mean with geometric SD. Non-parametric Mann Whitney U test (b-f) or Logrank (Mantel-Cox) test (h) was used to compare groups. ** p<0.01.

[0043] FIGS.17A–17I illustrate modulation of lipid to DNA ratio does not affect T cell responses. FIGS.17A–17C: Biophysical characterization of DNA-LNPs at different N / P ratios. (FIG.17A) Particle size (FIG.17B) Polydispersity index (PDI) (FIG.17C) Zeta potential. FIG. 17D–17I: Cytokine / marker expressing effector CD8+ and CD4+ T cells; CD107a+ CD8+ Tcells (FIG.17D), IFNγ+ CD8+ T cells (FIG.17E), TNFα+ CD8+ T cells (FIG.17F), IFNγ+ CD4+ T cells (FIG.17G), TNFα+ CD4+ T cells (FIG.17H), IL-2+ CD4+ T cells (FIG.17I). Cells pre-gated on live CD3+ CD4- CD8+ CD44+ CD62L- (FIGS. 17D–17F) or live CD4+ CD8- CD44+ CD62L- (FIGS.17G–17I). Dots represent individual animals; n=8-9 animals per group (FIGS. 17D–17I). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Related to FIGS.1A–1C.

[0044] FIGS.18A–18D illustrate Activation signature of CD4+ T cells and IFNγ+ NK cells 24 hours post immunization. FIG.18A: Representative FACS plots of CD69+ CD4+ T cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3+ NKp46-. FIG. 18B: Bar plot showing quantification of CD69+ CD4+ T cells as in (FIG.18A) in the popliteal DLN, iliac DLN, and spleen. FIG. 18C: Representative FACS plots of IFNv+ NK cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3- NKp46 +. FIG.18D: Bar plot showing quantification of IFNγ+ NK cells as in (FIG.18C) in the popliteal DLN, iliac DLN, and spleen. Dots represent individual animals; n=4-5 animals per group; Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups in (FIG.18B and 18D). * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Related to FIGS.2A–2C.

[0045] FIGS. 19A–19I illustrate Innate immune population frequency and activation signature in the spleen 24 hours post immunization. FIGS.19A–19H: Frequency of total rDCs (FIG. 19A), CD11b+ rDCs (FIG. 19B), CD8α+ rDCs (FIG. 19C), total mDCs (FIG. 19D), CD11b+ mDCs (FIG. 19E), CD103+ mDCs (FIG. 19F), neutrophils (FIG. 19G), and pDCs (FIG.19H) in the spleen expressed as a frequency of CD45+ cells. FIG.19I: Quantification of CD86 expression on all innate populations in FIGS. 19A–19H. Dots represent individual animals; n=10 animals per group. Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Related to FIGS.2A–2C.

[0046] FIGS. 20A–20E illustrate overlapping genes with active vaccination groups demonstrates IFN-associated gene signature. Upregulated and downregulated genes amongDNA-LNP, mRNA-LNP, and protein in adjuvant immunized mice relative to Naïve. FIG.20A: NK cells. FIG.20B: Neutrophils. FIG.20C: Monocytes. FIG.20D: pDC. FIG.20E: cDC.

[0047] FIGS.21A–21C illustrate dosing of DNA-LNP and mRNA-LNP elicits similar CD4+ T cell responses. FIGS.21A–21C: Frequency of activated CD4+ T cells expressing IFNγ (FIG. 21A), TNFα (FIG. 21B), or IL-2 (FIG.21C) after immunization with varying doses of HA DNA-LNP or mRNA-LNP as indicated. Pregated on live CD3+ CD8- CD4+ CD44+ CD62L-. Dots represent individual animals; n=5 animals per group. Plots show geometric mean with geometric SD. Related to Figure 4.

[0048] FIG.22 illustrates T and NK cell activation gating strategy. Gating strategy for activation of CD4+ T, CD8+ T, and NK cells.

[0049] FIGS.23A and 23B illustrate innate immune population gating strategy. FIGS. 23A and B: Gating strategies for innate immune populations in the DLN (FIG.23A) and spleen (FIG.23B).

[0050] FIGS.24A and 24B illustrate germinal center and T cell gating strategy. FIGS. 24A and 24B: Gating strategies for germinal center Tfh and GC B cells (FIG.24A) and T cell responses in the spleen (FIG.24B).

[0051] FIGS. 25A–25C illustrate pVAC2 expereiments. FIG. 25A illustrates an experiment outline with (1) pVAC2 DNA-LNP and empty LNPS, (2) CA09 HA mRNA-LNP and empty LNPs, and (3) CA09 HA mRNA-LNP and pVAC2 DNA-LNP administration. FIG. 25B illustrates pVAC2. FIG.25C illustrates the pVAC2 sequence and discloses SEQ ID NOS 8-10, respectively, in order of appearance.

[0052] FIGS. 26A–26C illustrates germinal center responses (Tfh, GC B cell, CA09 HA-specific, FIG.26A, 26B, and 26C, respectively).

[0053] FIGS. 27A and 27B illustrate T cell responses using ELISpot ( FIG. 27A), effector cytokine-secreting CD4+ T cell responses (top row, FIG.27B) and effector cytokine- secreting / marker-positive CD8+ T cell responses (bottom row, FIG.27B).

[0054] FIG. 28 illustrates an experimental outline with (1) CA09 HA protein in Addavax (1:1 v / v) (1 μg), (2) CA09 HA protein and empty LNPs (1 μg + 40 μg), and (3) CA09 HA protein and pVAX DNA-LNP (1 μg + 1 μg) administration.

[0055] FIGS. 29A–29C illustrate improved Tfh ( FIG. 29A) responses with pVAX DNA-LNP co-immunization with recombinant protein relative to eLNPs, and total GC B and antigen-specific GC B cell responses ( FIG.29B and FIG.29C, respectively) with recombinant HA when co-delivered with eLNPs or pVAX DNA-LNPs.

[0056] FIGS.30A and 30B illustrate HA-specific T cell responses using ELISpot (FIG. 30A) with pVAX DNA-LNPs, cytokine-secreting CD4+ T cell responses (top row, FIG.30B) and effector cytokine-secreting / marker-positive CD8+ T cell responses (bottom row, FIG. 30B).

[0057] FIG.31 illustrates and experimental outline with 1) HA DNA-LNP (2 μg), (2) HA mRNA-LNP (2 μg), and (3) HA DNA-LNP and HA mRNA-LNP (1 μg + 1 μg) administration.

[0058] FIG.32 illustrates the frequency among total lymphocytes (CD45+ cells) (FIG. 32, top row), and the mean fluorescence intensity of CD86 (FIG.32, bottom row).

[0059] FIG.33 illustrates an experimental outline of 1) pVAX DNA-LNP (1 μg), (2) HA DNA-LNP (1 μg), (3) HA mRNA-LNP (1 μg), and (4) HA DNA-LNP and HA mRNA- LNP (0.5 μg + 0.5 μg) administration.

[0060] FIGS. 34A–34C illustrate germinal center responses (Tfh, GC B cell, CA09 HA-specific, FIG.34A, 34B, and 34C, respectively).

[0061] FIGS.35A and 35B illustrate HA-specific T cell responses using ELISpot (FIG. 35A), effector cytokine-secreting CD4+ T cell responses (top row, FIG. 35B), and effector cytokine-secreting / marker-positive CD8+ T cell responses (bottom row, FIG.35B).

[0062] FIGS. 36A–36P show that evDNA-LNPs enhance rHA vaccine-induced humoral and T cell responses. (A) Schematic of immunization regimen. Mice were immunized with 1µg of CA09 rHA formulated with Addavax, eLNPs, or evDNA-LNPs. Humoral immune responses were measured longitudinally, and adaptive immune responses in the spleen were profiled on Day 12. Memory responses were surveyed at 63 days post immunization. (B) CA09 HA-specific total IgG responses in serum measured by ELISA. (C) HAI titers to CA09. (D) HAI titers to CA09 at week 4 post immunization. (E) IFNγ-secreting responses in the spleen at Day 12 measured by ELISpot. (F) Representative FACS plot of IFNγ expression in effectorCD4+T cells. (G-I) Frequency of effector (CD44+CD62L-) CD4+T cells expressing IFNγ (G), IL-2 (H), and TNFα (I). (J-L) Frequency of effector CD8+T cells expressing IFNγ (J), CD107a (K), and TNFα (L). (M) FACS plot showing CA09 HA-specific MBCs in the spleen. (N-O) Quantification of the frequency (N) and number (O) of CA09 HA-specific MBCs. (P) IFNγ- secreting responses in the spleen measured by ELISpot. Dots on bar graphs represent individual mice; data combined from two independent experiments, n=6-8 mice per group. One-way ANOVA adjusted for multiple comparisons used to compare groups. Two-way ANOVA adjusted for multiple comparisons used to compare groups and pools in (E, P). Graphs show geometric mean / SD (B-D) or mean / SD (E, G-L, N-P). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0063] FIGS. 37A–37) show that the adjuvant activity of evDNA-LNPs requires encapsulation of DNA within LNPs for T cell but not GC responses. (A) Schematic of immunization regimen. Mice were immunized with evDNA-LNPs or 1µg of CA09 rHA formulated with naked pVAX plasmid, eLNPs, eLNPs + pVAX plasmid, or evDNA-LNPs. GC and T cell responses were assessed in the DLNs and spleens respectively 14 days post immunization. (B) FACS plot showing activated Tfh responses. (C-D) Quantification of the frequency (C) and number (D) of activated Tfh responses. (E) FACS plot showing total GC B cell responses. (F-G) Quantification of the frequency (F) and number (G) of total GC B cells. (H) FACS plot showing CA09 HA-specific GC B cells. (I-J) Quantification of the frequency (I) and number (J) of CA09 HA-specific GC B cells. (K) IFNγ-secreting responses in the spleen measured by ELISpot. (L-N) Frequency of effector CD4+T cells expressing IFNγ (L), TNFα (M), and IL-2 (N). (O) Frequency of effector CD8+T cells expressing IFNγ. Dots represent individual mice; data from one independent experiment of n=3-5 mice. One-way ANOVA adjusted for multiple comparisons used to compare groups. Two-way ANOVA adjusted for multiple comparisons used to compare groups and pools in (K). Graphs show mean / SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0064] FIGS.38A–38M show that evDNA-LNPs improve challenge outcome in young and aged mice. (A) Schematic of immunization and challenge regimen. Young mice were immunized with the constructs indicated and challenged 14 days post immunization. (B)Average weight loss by group over time. (C) Survival curves. (D-G) Weight loss of individual vaccine groups evDNA-LNP (D), HA 0.5µg + Addavax (E), HA 0.5µg + evDNA-LNP (F), and HA 0.1µg + evDNA-LNP (G) showing individual mice. (H) Schematic of immunization and challenge regimen. Aged mice were immunized with the constructs indicated and challenged 14 days post immunization. (I) Average weight loss by group over time. (J) Survival curves. (K-M) Weight loss of individual vaccine groups evDNA-LNP (K), HA 1µg + Addavax (L), HA 1µg + evDNA-LNP (M) showing individual mice. Data from one independent experiment; n=6-10 mice per group. Black lines show the mean (D-G, K-M). Graphs show mean / SD. Log-rank test used to compare groups (C, J). ****p<0.0001.

[0065] FIGS.39A–39H show that evDNA-LNPs and antigen-expressing DNA-LNPs can synergize with licensed TIV or recombinant trivalent vaccine approaches. (A) Schematic of immunization regimen. Mice were immunized with 1µg (per HA) of TIV or co-immunized with evDNA-LNPs. Serology was assessed longitudinally. (B-D) Longitudinal serum IgG binding titers against H1N1 A / Victoria / 4897 / 2022 HA (B), H3N2 A / Massachusetts / 18 / 2022 HA (C), or Victoria B / Austria / 1359417 / 2021 HA (D) measured by ELISA. (E) Schematic of immunization regimen. Mice were immunized with 1µg (per HA) of recombinant H1N1 A / Wisconsin / 67 / 2022 HA (rH1), H3N2 A / Massachusetts / 18 / 2022 HA (rH3), and Victoria B / Austria / 1359417 / 2021 HA (rHB) with evDNA-LNPs or H5N1 A / Texas / 37 / 2024 HA DNA- LNP (H5 DNA-LNP). Humoral immune responses were assessed at week 4 post immunization. (F) Longitudinal serum IgG binding titers against H1N1 A / Wisconsin / 67 / 2022 HA, H3N2 A / Massachusetts / 18 / 2022 HA, Victoria B / Austria / 1359417 / 2021 HA, and H5N1 A / Texas / 37 / 2024 HA measured by ELISA. (G) Microneutralization (MNA) titer to a H5N1 A / bald eagle / FL / W22-134-OP / 2022 PR8-reassortant virus. (H) H5N1 A / Michigan / 90 / 2024 pseudovirus neutralization titers at 4 weeks post immunization. Dots represent individual mice; data from one independent experiment; n=5 mice per group. Graphs show geometric mean / SD. Non-parametric Mann-Whitney U test used to compare groups. **p<0.01.

[0066] FIGS.40A–40I show that evDNA-LNP adjuvant induces strong GC responses. (A) FACS plot showing activated Tfh responses. (B-C) Quantification of the frequency (B) and number (C) of activated Tfh responses. (D) FACS plot showing total GC B cell responses.(E-F) Quantification of the frequency (E) and number (F) of total GC B cells. (G) FACS plot showing CA09 HA-specific GC B cells. (H-I) Quantification of the frequency (H) and number (I) of CA09 HA-specific GC B cells. Dots represent individual mice; data from two (B, E, H) or one (C, F, I) independent experiment, n=3-6 mice per group. One-way ANOVA adjusted for multiple comparisons used to compare groups. Graphs show mean / SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0067] FIGS. 41A–41G show that evDNA-LNP adjuvant is dose sparing for recombinant protein and supports robust serology at 0.1µg of adjuvant. (A) Schematic of immunization regimen. Mice were immunized with de-escalating doses of HA (1µg, 0.5µg, 0.1µg) formulated in Addavax or with evDNA-LNPs. Serum antibody responses were assessed over time. (B) CA09 HA-specific serum IgG responses at 2 weeks post immunization. (C) CA09 HA-specific serum IgG response over time. (D) Timecourse of serum HAI titers against CA09. (E) Schematic of immunization regimen. Mice were immunized with 1µg HA formulated with either 1µg or 0.1µg of evDNA-LNPs. (F) Timecourse of CA09 HA-specific serum IgG responses. (G) Timecourse of serum HAI titers against CA09. Dots in bar graphs represent individual mice; data from one independent experiment, n=3-5 mice per group. Non- parametric Mann-Whitney U test used to compare groups (E-F). Graphs show geometric mean / SD. *p<0.05.

[0068] FIGS. 42A–42J show that evDNA-LNP adjuvant modulates innate immune populations. (A) Schematic of immunization regimen. Mice were left naïve immunized with 1µg of CA09 rHA formulated with Addavax, eLNPs, or evDNA-LNPs. Innate immune populations were assessed in the popliteal LN 24 hours post immunization. (B-D) Plots quantifying the frequency of total mDCs (B), CD11b+mDCs (C), and CD103+mDCs (D). (E) Quantification of CD86 expression on mDC populations gated as above. (F-I) Frequency of neutrophils (F), pDCs (G), classical monocytes (H), and non-classical monocytes (I) in the popliteal lymph node 24 hours post immunization. (J) Quantification of CD86 expression on innate immune populations. Mo = monocyte. Dots represent individual mice; data combined from two independent experiments; n=6-9 mice per group. Graphs show mean with SD. One-way (B-D, F-I) or two-way (E, J) ANOVA adjusted for multiple comparisons used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0069] FIGS.43A–43E show that co-formulation with evDNA-LNPs improve humoral immune responses in aged mice. (A) Schematic of immunization regimen. Mice were immunized with 1µg or 2.5µg of rHA formulated in Addavax or with evDNA-LNPs. Serology was monitored longitudinally. (B-C) CA09 HA-specific serum IgG responses after immunization with 1µg rHA (B) or 2.5µg rHA (C) formulated with either adjuvant. (D-E) HAI titers to CA09 after immunization with 1µg rHA (D) or 2.5µg rHA (E) formulated with either adjuvant. Dots represent individual mice; data from one independent experiments; n=4-5 mice per group. Graphs show geometric mean with SD. Two-way ANOVA used to compare groups. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0070] The present disclosure relates to lipid nanoparticles (LNP) as well as compositions thereof. In some embodiments, the compositions comprise at least one lipid of the present disclosure and at least one helper lipid. In some embodiments, the disclosure provides a composition comprising at least one lipid or LNP for delivery of DNA molecules.

[0071] In various embodiments, the disclosure relates to methods of vaccination using the composition comprising at least one lipid or LNP. In some embodiments, the disclosure provides a composition comprising at least one lipid or LNP for preventing or treating various diseases or disorders in a subject in need thereof. Definitions

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the exemplary methods and materials are described.

[0073] As used herein, each of the following terms has the meaning associated with it in this section.

[0074] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0075] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0076] “Alkyl” refers to a straight or branched hydrocarbon chain radical consisting solely of carbon and hydrogen atoms, which is saturated or unsaturated (i.e., contains one or more double and / or triple bonds), having from one to twenty-four carbon atoms (C1-C24 alkyl), one to twelve carbon atoms (C1-C12 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1-methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-1-enyl, pent- 1- enyl, penta-1,4-dienyl, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless specifically stated otherwise, an alkyl group is optionally substituted. The term “alkyl,” by itself or as part of another substituent means, unless otherwise stated, a straight or branched chain hydrocarbon having the number of carbon atoms designated (i.e., C1-6 means one to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups.

[0077] As used herein, the term “substituted alkyl” means alkyl, as defined above, substituted by one, two or three substituents selected from the group consisting of halogen, - OH, alkoxy, -NH2, -N(CH3)2, -C(=O)OH, trifluoromethyl, -C≡N, -C(=O)O(C1-C4)alkyl, - C(=O)NH2, -SO2NH2, -C(=NH)NH2, and -NO2, preferably containing one or two substituents selected from halogen, -OH, alkoxy, -NH2, trifluoromethyl, -N(CH3)2, and - C(=O)OH, more preferably selected from halogen, alkoxy and -OH. Examples of substituted alkyls include, but are not limited to, 2,2-difluoropropyl, 2-carboxycyclopentyl and 3- chloropropyl.

[0078] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely ofcarbon and hydrogen, which is saturated or unsaturated (i.e., contains one or more double (alkenylene) and / or triple bonds (alkynylene)), and having, for example, from one to twenty- four carbon atoms (C1-C24 alkylene), one to fifteen carbon atoms (C1-C15 alkylene),one to twelve carbon atoms (C1-C12 alkylene), one to eight carbon atoms (C1-C8 alkylene), one to six carbon atoms (C1-C6 alkylene), two to four carbon atoms (C2-C4 alkylene), one to two carbon atoms (C1-C2 alkylene), e.g., methylene, ethylene, propylene, n-butylene, ethenylene, propenylene, n-butenylene, propynylene, n-butynylene, and the like. The alkylene chain is attached to the rest of the molecule through a single or double bond and to the radical group through a single or double bond. The points of attachment of the alkylene chain to the rest of the molecule and to the radical group can be through one carbon or any two carbons within the chain. Unless stated otherwise specifically in the specification, an alkylene chain may be optionally substituted.

[0079] “Cycloalkyl” or “carbocyclic ring” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like.

[0080] Unless specifically stated otherwise, a cycloalkyl group is optionally substituted. “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0081] As used herein, the term “heteroalkyl” by itself or in combination with another term means, unless otherwise stated, a stable straight or branched chain alkyl group consisting of the stated number of carbon atoms and one or two heteroatoms selected from the group consisting of O, N, Si, P, and S, and wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) may be placed at any position of the heteroalkyl group, including between the rest of the heteroalkylgroup and the fragment to which it is attached, as well as attached to the most distal carbon atom in the heteroalkyl group. Examples include: -O-CH2-CH2-CH3, -CH2-CH2-CH2-OH, - CH2-CH2-NH-CH3, -CH2-S-CH2-CH3, and -CH2CH2-S(=O)-CH3. Up to two heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3, or -CH2-CH2-S-S-CH3.

[0082] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered non- aromatic ring radical which consists of two to twelve carbon atoms and from one to six heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur. Unless stated otherwise specifically in the specification, the heterocyclyl radical may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include fused or bridged ring systems; and the nitrogen, carbon or sulfur atoms in the heterocyclyl radical may be optionally oxidized; the nitrogen atom may be optionally quaternized; and the heterocyclyl radical may be partially or fully saturated. Examples of such heterocyclyl radicals include, but are not limited to, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2- oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4- piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 1-oxo-thiomorpholinyl, and 1,1-dioxo- thiomorpholinyl. Unless specifically stated otherwise, a heterocyclyl group may be optionally substituted.

[0083] As used herein, the term “aromatic” refers to a carbocycle or heterocycle with one or more polyunsaturated rings and having aromatic character, i.e. having (4n + 2) delocalized π (pi) electrons, where n is an integer.

[0084] As used herein, the term “aryl,” employed alone or in combination with other terms, means, unless otherwise stated, a carbocyclic aromatic system containing one or more rings (typically one, two or three rings) wherein such rings may be attached together in a pendent manner, such as a biphenyl, or may be fused, such as naphthalene. Examples include phenyl, anthracyl, and naphthyl. Preferred are phenyl and naphthyl, most preferred is phenyl

[0001] As used herein, the term “derivative” refers to of the compounds disclosed herein are pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms,isomers, solvates and combinations thereof. The “combinations” mentioned in this context are refer to derivatives falling within at least two of the groups: pharmaceutically acceptable salts, prodrugs, deuterated forms, radio-actively labeled forms, isomers, and solvates. Examples of radio-actively labeled forms include compounds labeled with tritium, phosphorous-32, iodine- 129, carbon-11, fluorine-18, and the like.

[0085] As used herein, the term “heteroaryl” or “heteroaromatic” refers to aryl groups which contain at least one heteroatom selected from N, O, Si, P, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. A polycyclic heteroaryl may include one or more rings that are partially saturated. Examples include tetrahydroquinoline, 2,3-dihydrobenzofuryl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2- imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3- isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2- thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2- quinoxalinyl, 5- quinoxalinyl, 3-quinolyl, and 6-quinolyl.

[0086] Examples of non-aromatic heterocycles include monocyclic groups such as aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine, pyrroline, imidazoline, pyrazolidine, dioxolane, sulfolane, 2,3-dihydrofuran, 2,5-dihydrofuran, tetrahydrofuran, thiophane, piperidine, 1,2,3,6-tetrahydropyridine, 1,4-dihydropyridine, piperazine, morpholine, thiomorpholine, pyran, 2,3-dihydropyran, tetrahydropyran, 1,4-dioxane, 1,3- dioxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-1,3-dioxepin and hexamethyleneoxide.

[0087] Examples of heteroaryl groups include pyridyl, pyrazinyl, pyrimidinyl (particularly 2- and 4-pyrimidinyl), pyridazinyl, thienyl, furyl, pyrrolyl (particularly 2- pyrrolyl), imidazolyl, thiazolyl, oxazolyl, pyrazolyl (particularly 3- and 5-pyrazolyl), isothiazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, 1,3,4-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3- oxadiazolyl, 1,3,4-thiadiazolyl and 1,3,4-oxadiazolyl.

[0088] Examples of polycyclic heterocycles include indolyl (particularly 3-, 4-, 5-, 6- and 7-indolyl), indolinyl, quinolyl, tetrahydroquinolyl, isoquinolyl (particularly 1- and 5- isoquinolyl), 1,2,3,4-tetrahydroisoquinolyl, cinnolinyl, quinoxalinyl (particularly 2- and 5- quinoxalinyl), quinazolinyl, phthalazinyl, 1,8-naphthyridinyl, 1,4-benzodioxanyl, coumarin, dihydrocoumarin, 1,5-naphthyridinyl, benzofuryl (particularly 3-, 4-, 5-, 6- and 7-benzofuryl), 2,3-dihydrobenzofuryl, 1,2-benzisoxazolyl, benzothienyl (particularly 3-, 4-, 5-, 6-, and 7- benzothienyl), benzoxazolyl, benzothiazolyl (particularly 2-benzothiazolyl and 5- benzothiazolyl), purinyl, benzimidazolyl (particularly 2-benzimidazolyl), benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl, pyrrolizidinyl, and quinolizidinyl.

[0089] The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.

[0090] As used herein, the term “amino aryl” refers to an aryl moiety which contains an amino moiety. Such amino moieties may include, but are not limited to primary amines, secondary amines, tertiary amines, masked amines, or protected amines. Such tertiary amines, masked amines, or protected amines may be converted to primary amine or secondary amine moieties. Additionally, the amine moiety may include an amine-like moiety which has similar chemical characteristics as amine moieties, including but not limited to chemical reactivity.

[0091] As used herein, the terms “alkoxy,” “alkylamino” and “alkylthio” are used in their conventional sense, and refer to alkyl groups linked to molecules via an oxygen atom, an amino group, a sulfur atom, respectively.

[0092] As used herein, the term “alkoxy” employed alone or in combination with other terms means, unless otherwise stated, an alkyl group having the designated number of carbon atoms, as defined above, connected to the rest of the molecule via an oxygen atom, such as, for example, methoxy, ethoxy, 1-propoxy, 2-propoxy (isopropoxy) and the higher homologs and isomers. Preferred are (C1-C3) alkoxy, particularly ethoxy and methoxy.

[0093] As used herein, the term “halo” or “halogen” alone or as part of another substituent means, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom, preferably, fluorine, chlorine, or bromine, more preferably, fluorine or chlorine.

[0094] The term “substituted” used herein means any of the above groups (e.g., alkyl, cycloalkyl or heterocyclyl) wherein at least one hydrogen atom is replaced by a bond to a non- hydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=O); hydroxyl groups (-OH); alkoxy groups (-ORa, where Ra is C1-C12 alkyl or cycloalkyl); carboxyl groups (-OC(=O)Ra or –C(=O)ORa, where Ra is H, C1-C12 alkyl or cycloalkyl); amine groups (-NRaRb, where Ra and Rb are each independently H, C1-C12 alkyl or cycloalkyl); C1-C12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkyl group. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as fluoro. In other embodiments, the substituent is a oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group. In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amine group.

[0095] As used herein, the term “nanoparticle” refers to particles having a particle size on the nanometer scale, less than 1 micrometer. For example, the nanoparticle may have a particle size up to about 50 nm. In another example, the nanoparticle may have a particle size up to about 10 nm. In another example, the nanoparticle may have a particle size up to about 6 nm. As used herein, “nanoparticle” refers to a number of nanoparticles, including, but not limited to, nanoclusters, nanovesicles, micelles, lamaellae shaped particles, polymersomes, dendrimers, and other nano-size particles of various other small fabrications that are known to those in the art.

[0096] The shapes and compositions of nanoparticles may be guided during condensation of atoms by selectively favoring growth of particular crystal facets to produce spheres, rods, wires, discs, cages, core-shell structures and many other shapes. The definitions and understandings of the entities falling within the scope of nanocapsule are known to those of skill in the art, and such definitions are incorporated herein by reference and for the purposes of understanding the general nature of the subject matter of the present application.

[0097] As used herein, “nucleic acid” is meant to include any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodiester linkages or modified linkages such as phosphotriester, phosphoramidate,siloxane, carbonate, carboxymethylester, acetamidate, carbamate, thioether, bridged phosphoramidate, bridged methylene phosphonate, phosphorothioate, methylphosphonate, phosphorodithioate, bridged phosphorothioate or sulfone linkages, and combinations of such linkages. The term nucleic acid also specifically includes nucleic acids composed of bases other than the five biologically occurring bases (adenine, guanine, thymine, cytosine, and uracil). The term “nucleic acid” typically refers to large polynucleotides.

[0098] “Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0099] An “isolated nucleic acid” refers to a nucleic acid segment or fragment, which has been separated from sequences which flank it in a naturally occurring state, i.e., a DNA fragment, which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.

[0100] The term also applies to nucleic acids which have been substantially purified from other components, which naturally accompany the nucleic acid, i.e., RNA or DNA or proteins, which naturally accompany it in the cell. The term therefore includes, for example, a recombinant DNA or RNA, which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA or RNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences. It also includes a recombinant DNA or RNA, which is part of a hybrid gene encoding additional polypeptide sequence.

[0101] The term “DNA” as used herein is defined as deoxyribonucleic acid.

[0102] The term “RNA” as used herein is defined as ribonucleic acid.

[0103] “Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequenceof nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0104] “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0105] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

[0106] The term “immune response” is used herein is meant to refer to the activation of a host's immune system, e.g., that of a mammal, in response to the introduction of nucleic acid molecules comprising a nucleotide sequence encoding neoantigens a described herein.

[0107] “Immunogen” refers to any substance introduced into the body in order to generate an immune response. That substance can a physical molecule, such as a protein, or can be encoded by a vector, such as DNA, mRNA, or a virus.

[0108] In the context of the present disclosure, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N- glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

[0109] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s).

[0110] Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell.

[0111] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0112] In certain instances, the polynucleotide or nucleic acid of the disclosure is a “ nucleic acid,” which refers to a nucleic acid comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification. For example, over onehundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[0113] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0114] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA or RNA methods.

[0115] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0116] The term “recombinant RNA” as used herein is defined as RNA produced by joining pieces of RNA from different sources.

[0117] The “percent identity” or "percent homology" of two polynucleotide or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (a part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, Calif.)) using its default parameters. "Identical" or "identity" as used herein in the context of two or more nucleic acids or amino acid sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs inboth sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) may be considered equivalent. Identity may be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0. Briefly, the BLAST algorithm, which stands for Basic Local Alignment Search Tool is suitable for determining sequence similarity. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length within a query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., 1997). These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension for the word hits in each direction are halted when: 1) the cumulative alignment score falls off by the quantity X from its maximum achieved value; 2) the cumulative score goes to zero or below, due to the accumulation of one or more negative- scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indicationof the probability by which a match between two nucleotide sequences would occur by chance. For example, a nucleic acid is considered similar to another if the smallest sum probability in comparison of the test nucleic acid to the other nucleic acid is less than about 1, less than about 0.1, less than about 0.01, and less than about 0.001.

[0118] As used herein, the term “identical” refers to two or more sequences or subsequences which are the same.

[0119] In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using a comparison algorithm or by manual alignment and visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. Such percentages to describe the “percent identity” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 sequential units in length, or, where not specified, across the entire sequence. This definition also refers to the complement of a test sequence.

[0120] “Variant” as the term is used herein, is a nucleic acid sequence or a peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence respectively, but retains essential biological properties of the reference molecule. Changes in the sequence of a nucleic acid variant may not alter the amino acid sequence of a peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions and truncations. Changes in the sequence of peptide variants are typically limited or conservative, so that the sequences of the reference peptide and the variant are closely similar overall and, in many regions, identical. A variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. A variant of a nucleic acid or peptide can be a naturally occurring, such as an allelic variant, or can be a variant that is not known to occur naturally. Non-naturally occurringvariants of nucleic acids and peptides may be made by mutagenesis techniques or by direct synthesis. In various embodiments, the variant sequence is at least 99%, at least 98%, at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, at least 92%, at least 91%, at least 90%, at least 89%, at least 88%, at least 87%, at least 86%, at least 85% identical to the reference sequence.

[0121] As used herein, “fragment” is defined as at least a portion of the variable region of the immunoglobulin molecule which binds to its target, i.e. the antigen binding region. Some of the constant region of the immunoglobulin may be included.

[0122] As used herein, the term “linkage” refers to bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and another molecule. Such bonds may include, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally hydrolyze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5′ hydroxyl group of an oligonucleotide.

[0123] The term “gene,” as used herein, refers to a nucleic acid molecule that encodes a protein or functional RNA (for example, a tRNA). A gene can include regions that do not encode the final protein or RNA product, such as 5′ or 3′ untranslated regions, introns, ribosome binding sites, promoter or enhancer regions, or other associated and / or regulatory sequence regions.

[0124] The terms “gene expression” and “expression” are used interchangeably herein to refer to the process by which inheritable information from a gene, such as a DNA sequence, is made into a functional gene product, such as protein or RNA.

[0125] As used herein, the terms “promoter” or “regulatory sequence” mean a nucleic acid sequence which is required for expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be the core promoter sequence and in other instances, this sequence may also include an enhancer sequence and other regulatory elements which are required for expression of the gene product. The promoter / regulatory sequence may, for example, be one which expresses the gene product in a tissue specific manner.

[0126] The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0127] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross- species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0128] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from abiological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0129] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[0130] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0131] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

[0132] The term “hyperproliferative disorder” refers to a disease or disorder characterized by abnormal proliferation, abnormal growth, abnormal senescence, abnormal quiescence, or abnormal removal of cells in an organism, and includes all forms of hyperplasia, neoplasia, and cancer. In some embodiments, a hyperproliferative disease is a cancer derived from the gastrointestinal tract or urinary system. In some embodiments, a hyperproliferative disease is a cancer of the adrenal gland, bladder, bone, bone marrow, brain, spine, breast, cervix, gall bladder, ganglia, gastrointestinal tract, stomach, colon, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, or uterus. In some embodiments, a hyperproliferative disease is a cancer chosen from: lung cancer, bone cancer, CMML, pancreatic cancer, skin cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, testicular, gynecologic tumors (e.g., uterine sarcomas, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina or carcinoma of the vulva), Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system (e.g., cancer of the thyroid, parathyroid or adrenal glands), sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidneyor ureter (e.g., renal cell carcinoma, carcinoma of the renal pelvis), or neoplasms of the central nervous system (e.g., primary CNS lymphoma, spinal axis tumors, brain stem gliomas or pituitary adenomas).

[0133] “Cancer,” as used herein, refers to the abnormal growth or division of cells.

[0134] Generally, the growth and / or life span of a cancer cell exceeds, and is not coordinated with, that of the normal cells and tissues around it. Cancers may be benign, pre- malignant or malignant. Cancer occurs in a variety of cells and tissues, including the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gall bladder, pancreas, etc.), respiratory system (e.g., larynx, lung, bronchus, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, genital system, (e.g., uterus, ovary, prostate, testis, etc.), urinary system (e.g., bladder, kidney, ureter, etc.), eye, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0135] An “effective amount” as used herein, means an amount that is therapeutically effective, prophylactically effective, or both.

[0136] The term “therapeutic” as used herein means a treatment. The term “prophylactic” as used herein means prevention. To be “therapeutically effective” means suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disordered state. To be prophylactically effective means preventing a disease or disordered state.

[0137] The term “therapeutically effective amount” refers to the amount of the subject compound or composition that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound or composition that, when administered, is sufficient to alleviate to some extent, one or more of the signs or symptoms of the disease or disordered state being treated. The therapeuticallyeffective amount will vary depending on the compound or composition, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0138] The term “prophylactically effective amount” refers to the amount of the subject compound or composition that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician in preventing a disease or disordered state. The term “prophylactically effective amount” includes that amount of a compound or composition that, when administered, is sufficient to prevent development of to some extent, one or more of the signs or symptoms of a disease or disordered state. The prophylactically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0139] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof or any multicellular organism, or cells thereof, whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human. In certain non-limiting embodiments, the patient, subject or individual is a fetus. In certain non-limiting embodiments, the patient, subject or individual is an embryo.

[0140] By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and / or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and / or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, preferably, a human.

[0141] To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0142] The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0143] The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

[0144] A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

[0145] “Optional” or “optionally” (e.g., optionally substituted) means that the subsequently described event of circumstances may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not. For example, “optionally substituted alkyl” means that the alkyl radical may or may not be substituted and that the description includes both substituted alkyl radicals and alkyl radicals having no substitution.

[0146] Ranges: throughout this disclosure, various aspects of the disclosure can be 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 disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0147] The present disclosure relates, in part, lipid nanoparticles (LNP) as well as compositions thereof, and methods of use thereof to deliver DNA molecules. This disclosure also relates, in part, to the discovery that said LNP, and / or compositions thereof, comprsing DNA are effective adjuvants for vaccines. Thus, in some aspects, the present disclosure also relates to the method of delivering DNA molecules using the LNP compositions. In various embodiments, the disclosure relates to methods of vaccination using the composition comprising an LNP. In some embodiments, the disclosure provides methods of preventing or treating diseases or disorders in a subject in need thereof using the composition comprising at least LNP for delivery of a DNA molecule along with a vaccine. The vaccine, in some embodiments comprises a nucleic acid encoding an antigen for inducing an immune response against an antigen associated with the disease or disorder.

[0148] Lipid Nanoparticles (LNP)

[0149] The present disclosure relates, in part, to lipid nanoparticle compositions (LNP) comprising a combination of one or more lipid compounds. In one embodiment, the LNP comprises at least one ionizable lipid compound.

[0150] The term “lipid nanoparticle” or “LNP” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which includes one or more lipids, for example a combination of a cationic or ionizable lipid, a helper lipid, a structural lipid (e.g., cholesterol) and a stability lipid.

[0151] In some embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm.

[0152] In some embodiments, the lipid nanoparticle comprises a DNA-LNP with a lipid to DNA weight ratio in the range of about 5:1 to about 50:1. In some embodiments, thelipid nanoparticle comprises a DNA-LNP with a lipid to DNA weight ratio in the range of about 10:1 to about 40:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 40:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 20:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 10:1.

[0153] In some embodiments, the lipid nanoparticle encapsulates a DNA cargo molecule. In some embodiments, the DNA cargo molecule is encapsulated within the lipid nanoparticle with an efficiency of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99%. In some embodiments, the lipid nanoparticle encapsulating a DNA cargo molecule is generated according to the method of mixing DNA diluted in citrate buffer (citrate buffer 50 mM, pH-4) at a concentration of 129 µg / mL with lipid containing ethanol at a volumetric ratio of 1:3 (ethanol: citrate buffer) using microfluidic mixing device.

[0154] In various embodiments, the lipids or the LNP of the present disclosure are substantially non-toxic.

[0155] For example, in some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 1 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 2 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 5 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 5.5 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 10 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 12 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 15 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 20 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 25 mol%. In some embodiments, the LNP comprises one or more lipidsof the present disclosure in a concentration of about 30 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 35 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 37 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 40 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 45 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 50 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 60 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 70 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 80 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 90 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 95 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 95.5 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 99 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 99.9 mol%. In some embodiments, the LNP comprises one or more lipids of the present disclosure in a concentration of about 100 mol%.

[0156] In various embodiments, the LNP comprises one or more ionizable or cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In some embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

[0157] In some embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, SM-102 or other lipids as described in Sabnis, et al,.2018,Mol Ther. 26(6):1509-1519, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N- (2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP); 3-(N-(N′,N′-dimethylaminoethane)- carbamoyl)cholesterol (DC-Chol), N-(1- (2,3-dioleoyloxy)propyl)-N-2- (sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), 1,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(1,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present disclosure. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3- phosphoethanolamine (DOPE), from GIBCO / BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO / BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.).

[0158] In some embodiments, the cationic lipid is an amino lipid. Exemplary amino lipids useful in the disclosure include those described in WO 2012 / 016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin- TAP.Cl), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N- dilinoleylamino)-1,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2- dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA).

[0159] In various embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 0.1 mol% to about 100 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 1 mol% to about 100 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 10 mol% to about 70 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 10 mol% to about 50 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 15 mol% to about 45 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 35 mol% to about 40 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 40 mol% to about 45 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure in a concentration range of about 45 mol% to about 50 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 41 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 42 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 43 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 44 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 45 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 46 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 47 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 48 mol%. In some embodiments, the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 49 mol%. In some embodiments,the LNP comprises one or more ionizable or cationic lipid of the present disclosure at about 50 mol%.

[0160] In various embodiments, the LNP further comprises at least one helper compound. In some embodiments, the helper compound is a helper lipid, helper polymer, or any combination thereof. In some embodiments, the helper lipid is phospholipid, cholesterol lipid, polymer, cationic lipid, neutral lipid, charged lipid, steroid, steroid analogue, polymer conjugated lipid, stabilizing lipid, or any combination thereof.

[0161] In various embodiments, the LNP comprises one or more helper compound in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.01 mol% to about 99.9 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.1 mol% to about 90 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.1 mol% to about 70 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 5 mol% to about 95 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.5 mol% to about 50 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 0.5 mol% to about 47 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration range of about 2.5 mol% to about 47 mol%.

[0162] For example, in some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.01 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.1 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 0.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 1 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 1.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 2 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 2.5 mol%. In someembodiments, the LNP comprises one or more helper compound in a concentration of about 5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 10 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 12 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 15 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 16 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 20 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 25 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 30 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 35 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 37 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 40 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 45 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 46.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 47 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 50 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 60 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 63 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 70 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 80 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 90 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 95 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 95.5 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 99 mol%. In some embodiments, the LNP comprises one or more helper compound in a concentration of about 100 mol%.

[0163] In some embodiments, the phospholipid is dioleoyl-phosphatidylethanolamine (DOPE) or a derivative thereof, distearoylphosphatidylcholine (DSPC) or a derivative thereof, distearoyl-phosphatidylethanolamine (DSPE) or a derivative thereof, stearoyloleoylphosphatidylcholine (SOPC) or a derivative thereof, 1-stearioyl-2-oleoyl- phosphatidyethanol amine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTAP) or a derivative thereof, or any combination thereof.

[0164] For example, in some embodiments, the LNP comprises a phospholipid in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 15 mol% to about 50 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 10 mol% to about 40 mol%. In some embodiments, the LNP comprises a phospholipid in a concentration range of about 16 mol% to about 40 mol%.

[0165] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof.

[0166] For example, in some embodiments, the LNP comprises a cholesterol lipid in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a cholesterol lipid in a concentration range of about 20 mol% to about 50 mol%. In some embodiments, the LNP comprises a cholesterol lipid in a concentration range of about 20 mol% to about 47 mol%. In some embodiments, the LNP comprises a cholesterol lipid in a concentration of about 47 mol% and DOPE in a concentration of about 16 mol%.

[0167] In some embodiments, the polymer is polyethylene glycol (PEG) or a derivative thereof. For example, in some embodiments, the LNP comprises a polymer in a concentration range of about 0 mol% to about 100 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 10 mol%. In some embodiments, the LNP comprises a polymer in a concentration range of about 0.5 mol% to about 2.5 mol%.

[0168] In some embodiments, the LNP comprises one or more neutral lipid. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0169] Exemplary neutral lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), distearoyl-phosphatidylethanolamine (DSPE)-maleimide- PEG, distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), stearoyloleoylphosphatidylcholine (SOPC), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In one embodiment, the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

[0170] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0171] In some embodiments, the LNP comprises one or more steroid. A “steroid” is a compound comprising the following carbon skeleton:.

[0172] In some embodiments, the steroid or steroid analogue is cholesterol.

[0173] In some embodiments, the LNP comprises one or more anionic lipid. The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N- dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0174] In some embodiments, the LNP comprises one or more polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[0175] In some embodiments, the LNP comprises an additional, stabilizing-lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

[0176] In some embodiments, one or more additional lipid (in addition to a cationic or ionizable lipid) is present in the LNP in an amount from about 1 mol% to about 10 mol%. In one embodiment, the additional lipid is present in the LNP in an amount from about 1 mol% to about 5 mol%. In one embodiment, one or more additional lipid is present in the LNP in about 1 mol% or about 2.5 mol%.

[0177] In various embodiments, the lipids or the LNPs described herein readily transport to a tissue of interest. For example, in various embodiments, the lipids or the LNPs described herein readily transport through a cell membrane to a cell. In various embodiments,the lipids or the LNP described herein efficiently transport through a cell membrane and a nuclear membrane to deliver a DNA molecule to a nucleus of a cell. In some embodiments, the lipids or the LNP described herein transport through both the cell membrane and the nuclear membrane with enhanced efficacy.

[0178] In some embodiments, the LNP comprises a combination of the ionizable cationic lipid of SM-102, a helper lipid comprising DSPC, cholesterol and MG-PEG-2000. In some embodiments, the LNP comprises a ratio of 50 (SM-102) to 38.5 (Cholesterol) to 10 (DSPC) to 1.5 (DMG-PEG-2000).

[0179] In some embodiments, a derivative of SM-102 is a compound of Formula I:Formula I wherein n is from about 1 to about 3; and wherein R1is an alkyl or heteroalkyl group comprising from about 7 to about 25 carbon atoms. In some embodiments, R1comprises a total of from about 7 to about 25 carbon atoms, wherein at least one of the carbon atoms forms a carboxyl group between two contiguous hydrocarbon chains. In some embodiments n is 1 or 2, and R1comprises two non-contiguous alkyl or heteroalkyl groups covalently bound to a COO group. In some embodiments n is 1 or 2, and R1 comprises a first and a second non- contiguous alkyl or heteroalkyl chains covalently bound to a COO, wherein the first alkyl chain is from about 3 to about 5 contiguously bound carbon atoms in length and the second alkyl chain is from about 2 to about 14 carbon atoms in length. In some embodiments, n is 1, 2 or 3, and R1 is a C12 to C28 monoglyceride, alkenyl, alkyl, aryl, or aralkyl.

[0180] In some embodiments, the LNP comprises a derivative of SM-102, and optionally at the ratio in the compositions disclosed herein for SM-102. In some embodiments,the SM-102 derivative is a compound having Formula I, wherein R1 is chosen from one of the following:

[0181] In some embodiments, the SM-102 derivative is a compound having Formula I, wherein R1is chosen from one of the following:

[0182] In some embodiments, the SM-102 derivative is a compound having Formula I, wherein R1is chosen from one of the following:

[0183] In some embodiments, the composition comprises one or a plurality of molecules covalently or non-covalently bound to the outside of the lipid LNP and the one or plurality of molecules is a targeting moiety, capable of targeting the LNP to a cell, cell population, tissue of interest, or any combination thereof. For example, in one embodiment, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

[0184] In some embodiments, the composition comprises one or more internalization domains. For example, in one embodiment, the composition comprises one or more domainswhich bind to a cell to induce the internalization of the LNP. For example, in one embodiment, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP.

[0185] In some embodiments, the LNP comprises an LNP disclosed in U.S. Pre-Grant Publication No. 2024 / 0002127, which published January 4, 2024, is titled “Cornonavirus Vaccine,” and is incorporated herein by reference in its entirety.

[0186] In some embodiments, the LNP comprises ((4- hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide, 1,2-Distearoyl-sn-glycero-3-phosphocholine, and cholesterol.

[0187] In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a steroid, and a polymer conjugated lipid. In some embodiments, the cationic lipid is ALC-0315 (described above), the neutral lipid is DSPC, the steroid is cholesterol, and the polymer conjugated lipid is ALC-0159.

[0188] In some embodiments, the LNP comprises one or more of the components shown in one, two or all of Tables A through C. In some embodiments, the LNP comprises all of the components shown in Table A, Table B, or Table C. In some embodiments, the LNP comprises components shown in Table A, Table B, or Table C at the proportions or concentrations shown in Table A, Table B, or Table C. Table ATable BTable C

[0189] [1] ALC-0315 is ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2- hexyldecanoate) / 6-[N-6-(2-hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2- hexyldecanoate.

[0190] [2] ALC-0159 is 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2- [2-(ω-methoxy(polyethyleneglycol2000)ethoxy]-N,N-ditetradecylacetamide.

[0191] [3] DSPC is 1,2-Distearoyl-sn-glycero-3-phosphocholine.

[0192] ALC-0315 has the following structure:Formula II

[0193] ALC-0159 has the following structure:Formula III

[0194] Cholesterol has the following structure:Formula IV

[0195] In some embodiments, the LNP comprises an LNP disclosed in U.S. Patent No. 8,598,139, which issued December 3, 2013, is titled “Lipid formulated dsRNA targeting the PCSK9 gene,” and is incorporated herein by reference in its entirety.

[0196] In some embodiments, the LNP comprises an LNP disclosed in U.S. Patent No. 8,809,292, which issued August 19, 2014, is titled “Compositions and Methods for Inhibiting Expression of the PCSK9 Gene,” and is incorporated herein by reference in its entirety.

[0197] In some embodiments, the LNP comprises an LNP disclosed in U.S. Pre-Grant Publication No 2024 / 0358652, which published January 24, 2024, is titled “Lipid Nanoparticle Formulations,” and is incorporated herein by reference in its entirety.

[0198] In some embodiments, the LNP comprises (a) a cationic and / or ionizable lipid; (b) a phospholipid; (c) a structural lipid; and (d) a PEGylated lipid. In some embodiments, (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the nanoparticle. In some embodiments, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the nanoparticle. In some embodiments, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the nanoparticle. In some embodiments, (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the nanoparticle. In some embodiments, (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the nanoparticle, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the nanoparticle, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the nanoparticle, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the nanoparticle.

[0199] (a) a cationic and / or ionizable lipid

[0200] In some embodiments, the cationic lipid of comprise the following structure:Formula V, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation. In some embodiments, R3and R4are both the same. In some embodiments, R3and R4are both linoleyl (C18), etc. In some embodiments, both R3and R4comprise at least two sites of unsaturation. In some embodiments, R3and R4are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In some embodiments, R3and R4are both linoleyl. In some embodiments, R3and R4comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl,linolenyl, and icosatrienyl. In some embodiments, the cationic lipid of Formula V is 1,2- dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA) or 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA).

[0201] In some embodiments, the cationic lipid comprises the following structure:Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation. In some embodiments, R3and R4are both the same. In some embodiments, R3and R4are both linoleyl (C18), etc. In some embodiments, R3and R4are different, i.e., R3is tetradectrienyl (C14) and R4is linoleyl (C18). In some embodiments, the cationic lipid is symmetrical, i.e., R3and R4are both the same. In some embodiments, both R3and R4comprise at least two sites of unsaturation. In some embodiments, R3and R4are independently selected from the group consisting of dodecadienyl, tetradecadienyl, hexadecadienyl, linoleyl, and icosadienyl. In some embodiments, R3and R4are both linoleyl. In some embodiments, R3and R4comprise at least three sites of unsaturation and are independently selected from, e.g., dodecatrienyl, tetradectrienyl, hexadecatrienyl, linolenyl, and icosatrienyl.

[0202] In some embodiments, the cationic lipid comprises the following structure:wherein R1and R2are either the same or different and independently optionally substituted C12- C24 alkyl, optionally substituted C12-C24 alkenyl, optionally substituted C12-C24 alkynyl, or optionally substituted C12-C24acyl; R3and R4are either the same or different and independently optionally substituted C1-C6 alkyl, optionally substituted C1-C6 alkenyl, or optionally substituted C1-C5 alkynyl or R3and R4may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6alkyl to provide a quaternary amine; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are either the same or different and independently O, S, or NH.

[0203] In some embodiments, the cationic lipid of Formula VII is 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3- dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-dilinoleyl-4-(4- dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5- dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino- [1,3]-dioxolane (DLin-K-MPZ), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2- dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyoxy-3- morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3- dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin- TAP.C1), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N- dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), or mixtures thereof. In some embodiments, the cationic lipid of Formula VII is DLin-K-C2-DMA (XTC2).

[0204] In some embodiments, the cationic lipid is DODAP, DLin-DMA, DLin-K- DMA, DLin-K2-DMA DLin-MC3-DMA.

[0205] In some embodiments, the cationic lipid comprises from about 40 mol % to about 60 mol %, from about 40 mol % to about 55 mol %, from about 40 mol % to about 50 mol %, from about 40 mol % to about 45 mol %, from about 45 mol % to about 60 mol %, from about 50 mol % to about 60 mol %, or from about 55 mol % to about 60 mol % of the total lipid present in the particle.

[0206] In some embodiments, the cationic lipid comprises about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 mol % of the total lipid present in the particle.

[0207] (b) a phospholipid

[0208] As used herein, a “phospholipid” is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (e.g., double or triple) bonds (e.g., one or more unsaturations). Particular phospholipids may facilitate fusion to a membrane. For example, a cationic phospholipid may interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane may allow one or more elements of a lipid-containing composition to pass through the membrane permitting, e.g., delivery of the one or more elements to a cell.

[0209] The lipid component of a lipid nanoparticle or composition may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids may assemble into one or more lipid bilayers. In general, phospholipids may include a phospholipid moiety and one or more fatty acid moieties. For example, a phospholipid may be a lipid according to Formula VIII:Formula VIII

[0210] Formula VIII represents a phospholipid moiety and R and R′ represent fatty acid moieties with or without unsaturation that may be the same or different. In some embodiments, the phospholipid moiety is phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, or a sphingomyelin. In some embodiments, the fatty acid moiety is lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha- linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, or docosahexaenoic acid.

[0211] Non-natural species including natural species with modifications and substitutions including branch-ing, oxidation, cyclization, and alkynes are also contemplated. In some embodiments, a phospholipid is functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group may undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions may be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0212] In some embodiments, the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof. In some embodiments, the phospholipid comprises diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids. In some embodiments, the acyl groups in these lipids are acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0213] In some embodiments, the LNP comprises distearoylphosphatidylcholine (DSPC). In some embodiments, the LNP comprises 1,2-dioleoyl-sn- glycerophosphoethanolamine (DOPE). In some embodiments, the LNP comprises both DSPC and DOPE.

[0214] (c) a structural lipid

[0215] The lipid component of an LNP may comprise one or more structural lipids. In some embodiments, the one or more structural lipids comprise one or more of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the one or more structural lipid comprises cholesterol. In some embodiments, the one or more structural lipid comprises cholesterol and one or more corticosteroid. In some embodiments, the one or more corticosteroid comprises prednisolone, dexamethasone, prednisone, or hydrocortisone, or a combination of two or more thereof. In some embodiments, the one or more structural lipid comprises squalene.

[0216] In some embodiments, the structural lipid comprises lipids containing geranyl acetate, farnesyl acetate or geranyl-geranyl, or ether, ester, or other derivatives.

[0217] In some embodiments, the structural lipid comprises from about 30 mol % to about 50 mol %, from about 30 mol % to about 45 mol %, from about 30 mol % to about 40 mol %, from about 30 mol % to about 35 mol %, from about 35 mol % to about 50 mol %, from about mol % to about 50 mol %, or from about 45 mol % to about 50 mol % of the total lipid present in the particle.

[0218] In some embodiments, the structural lipid comprises from 30 mol % to 50 mol %, from 30 mol % to 45 mol %, from 30 mol % to 40 mol %, from 30 mol % to 35 mol %,from 35 mol % to 50 mol %, from 40 mol % to 50 mol %, or from 45 mol % to 50 mol % of the total lipid present in the particle.

[0219] (d) a PEGylated lipid

[0220] In some embodiments, the lipid component of a lipid nanoparticle comprises one or more PEG or PEG-modified lipids. Such species may be alternately referred to as PEG Lipids or PEGylated lipids. As used herein, a “PEG lipid” or “PEGylated lipid” refers to a lipid comprising a polyethylene glycol component. In some embodiments, a PEG lipid comprises PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. In some embodiments, a PEG lipid comprises a PEG- c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0221] In some embodiments, the PEGylated lipid comprises 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000, also known as DMG-PEG.

[0222] In some embodiments, the PEGylated lipid comprises 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide (ALC-0159).

[0223] In some embodiments, the PEGylated lipid comprises a PEG component that has a molecular weight of any molecular mass as practically desired, including but not limited to, from about 100 Daltons (Da) to 10,000 Da or more as desired (including but not limited to, sometimes 0.1-10 kDa). The molecular weight of PEG may be of a wide range, including but not limited to, between about 100 Da and about 10,000 Da or more. PEG may be between about 100 Da and about 100,000 Da, including but not limited to 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, PEG is between about 100 Da and 10,000 Da, between about 1000 Da and 9,000 Da, between about 1000 Da and 8,000 Da, between about 1000 Da and 7,000 Da, between about 1000 Da and 6,000 Da, between about 1000 Da and 5,000 Da, between about 1000 Da and 4,000 Da, between about 1000 Da and 3,000 Da, or between about 1000 Da and 2,000 Da. In some embodiments, PEG is between about 1,000 Da and 5,000 Da. In some embodiments, PEG is between about 2,000 Da and 5,000 Da.

[0224] In some embodiments, the PEGylated lipid comprises from about 0.05 mol % to about 0.5 mol %, from about 0.06 mol % to about 0.5 mol %, from about 0.07 mol % to about 0.5 mol %, from about 0.08 mol % to about 0.5 mol %, from about 0.09 mol % to about 0.5 mol %, from about 0.1 mol % to about 0.5 mol %, from about 0.15 mol % to about 0.5 mol %, from about 0.2 mol % to about 0.5 mol %, from about 0.25 mol % to about 0.5 mol %, from about 0.3 mol % to about 0.5 mol %, from about 0.3 mol % to about 0.5 mol %, from about 0.35 mol % to about 0.5 mol %, from about 0.4 mol % to about 0.5 mol %, from about 0.45 mol % to about 0.5 mol %, from about 0.05 mol % to about 0.45 mol %, from about 0.05 mol % to about 0.4 mol %, from about 0.05 mol % to about 0.35 mol %, from about 0.05 mol % to about 0.3 mol %, from about 0.05 mol % to about 0.25 mol %, from about 0.05 mol % to about 0.2 mol %, from about 0.05 mol % to about 0.15 mol %, from about 0.05 mol % to about 0.1 mol %, from about 0.05 mol % to about 0.09 mol %, from about 0.05 mol % to about 0.08 mol %, from about 0.05 mol % to about 0.07 mol %, or from about 0.05 mol % to about 0.06 mol % of the total lipid present in the particle.

[0225] In some embodiments, the PEGylated lipid comprises from 0.05 mol % to 0.5 mol %, from 0.06 mol % to 0.5 mol %, from 0.07 mol % to 0.5 mol %, from 0.08 mol % to 0.5 mol %, from 0.09 mol % to 0.5 mol %, from 0.1 mol % to 0.5 mol %, from 0.15 mol % to 0.5 mol %, from 0.2 mol % to 0.5 mol %, from 0.25 mol % to 0.5 mol %, from 0.3 mol % to 0.5 mol %, from 0.3 mol % to 0.5 mol %, from 0.35 mol % to 0.5 mol %, from 0.4 mol % to 0.5 mol %, from 0.45 mol % to 0.5 mol %, from 0.05 mol % to 0.45 mol %, from 0.05 mol % to 0.4 mol %, from 0.05 mol % to 0.35 mol %, from 0.05 mol % to 0.3 mol %, from 0.05 mol % to 0.25 mol %, from 0.05 mol % to 0.2 mol %, from 0.05 mol % to 0.15 mol %, from 0.05 mol % to 0.1 mol %, from 0.05 mol % to 0.09 mol %, from 0.05 mol % to 0.08 mol %, from 0.05 mol % to 0.07 mol %, or from 0.05 mol % to 0.06 mol % of the total lipid present in the particle.

[0226] In some embodiments, the PEGylated lipid comprises 0.05 mol %, 0.06 mol %, 0.07 mol %, 0.08 mol %, 0.09 mol %, 0.1 mol %, 0.15 mol %, 0.2 mol %, 0.25 mol %, 0.3 mol %, 0.35 mol %, 0.4 mol %, or 0.45 mol % of the total lipid present in the particle.

[0227] In some embodiments, an LNP herein is as described above with respect to any one of Formulas I through VII but with the replacement of or addition to the lipid of one of Formulas I through VII with one or more of Formulas IX through XIV:Formula XII (2-(dioctadecylamino)ethyl 4-(dimethylamino)butanoateFormula XIV (Lipid C24)

[0228] LNP Compositions

[0229] In various aspects, the present disclosure also provides compositions comprising an LNP described herein. In various embodiments, the composition comprises one or more nucleic acid molecules. In some embodiments, one or more of the one or more nucleic acid molecules are encapsulated within the LNP. Various compositions may comprise more than one LNP, sometimes referred to as a first, second, third or the like LNP. Each of the first, second, third or the like LNPs may be independently selected from any LNP herein.

[0230] In one embodiment, the nucleic acid molecule is a DNA molecule. Examples of such nucleic acid include, but are not limited to: cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, cosmid, rolling circle amplified DNA product, artificial chromosomes, replicating DNA or any combination thereof. In some embodiments, the disclosure relates to a composition comprising one or a plurality of LNPs, at least about 1 of the LNPs encapsulates a nucleic acid molecule that is a DNA molecule. In some embodiments, the DNA molecule is a double-stranded DNA molecule. In some embodiments, the total amount of DNA the composition is from about 30 micrograms to about 100 micrograms encapsulated in an LNP with one of the disclosed molar ratios disclosed herein. In some embodiments, the total amount of DNA the composition is from about 3 micrograms to about 100 micrograms. In some embodiments, the total amount of DNA the composition is from about 3 micrograms to about 2 milligrams. In some embodiments, the total amount of DNA the composition is from about 1 milligrams to about 3 milligrams. In some embodiments, the total amount of DNA the composition is from about 0.1 milligrams to about 3 milligrams. In some embodiments, the total amount of DNA the composition is from about 0.03 milligrams to about 3 milligrams. In some embodiments, the total amount of DNA the composition is from about 0.03 milligrams to about 2 milligrams. In some embodiments, the total amount of DNA the composition is from about 0.05 milligrams to about 5 milligrams. In some embodiments, the total amount of DNA the composition is from about 3 micrograms to about 100 micrograms.

[0231] The disclosure also relates to a composition comprising one or a plurality of LNPs, wherein, if the composition comprises a plurality of LNPs, greater than about 40% ofthe LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 45% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 50% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 55% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 60% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 65% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 70% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 75% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 80% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 85% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 90% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 95% of the LNPs comprise one or more DNA molecules. In some embodiments, the composition comprises a plurality of LNPs and greater than about 98% of the LNPs comprise one or more DNA molecules.

[0232] The disclosure also relates to a composition comprising one or a plurality of LNPs, wherein, if the composition comprises a plurality of LNPs, greater than about 40% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 45% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 50% of theLNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 55% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 60% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 65% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 70% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 75% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 80% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 85% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 90% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosed molar ratios of lipid to cholesterol or derivatives thereof, and greater than about 95% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP. In some embodiments, the composition comprises a plurality of LNPs with any one of the disclosedmolar ratios of lipid to cholesterol or derivatives thereof, and greater than about 98% of the LNPs comprise about 2, 3, 4, 5, 6, 7, 8, 9, or about 10 DNA molecules per LNP.

[0233] The disclosure also relates to a composition comprising one or a plurality of LNPs, wherein at least about one or all of the LNPs has a zeta potential of from about -2 millivolts to about -22 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -25 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -20 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -30 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -15 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -10 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -9 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -8 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -7 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -2 millivolts to about -6 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -25 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -10 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -9 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -8 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -7 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -1 millivolts to about -6 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -0.5 millivolts to about -10 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -0.5 millivolts to about -9 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -0.5 millivolts to about -8 millivolts. In some embodiments, the one or all of the LNPs has a zeta potential of from about -0.5 millivolts to about -7 millivolts.In some embodiments, the one or all of the LNPs has a zeta potential of from about -0.5 millivolts to about -6 millivolts.

[0234] The disclosure also relates to a composition comprising one population or a plurality of LNPs wherein the population of lipid nanoparticles have an average diameter of from about 70 to about 77 nanometers. In some embodiments, the population of lipid nanoparticles have an average diameter of from about 65 to about 90 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 90 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 85 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 84 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 83 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 82 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 81 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 80 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 79 nanometers. the population of lipid nanoparticles have an average diameter of from about 70 to about 78 nanometers. the population of lipid nanoparticles have an average diameter of from about 71 to about 77 nanometers. the population of lipid nanoparticles have an average diameter of from about 72 to about 77 nanometers.

[0235] In various embodiments, the nucleic acid molecule comprises a nucleotide sequence encoding a therapeutic agent. Therapeutic agents that can be delivered using a DNA- LNP of the disclosure include, but are not limited to, cytokines, chemokines, costimulatory molecules, enzymes, antibodies, antigens, or any combination thereof.

[0236] In some embodiments, the DNA molecule comprises a promoter or regulatory sequence. In one embodiment, the DNA molecule comprises a promoter or regulatory sequence such that the DNA molecule is capable of directing expression of one or more encoded therapeutic agent. Thus, in one embodiment, the DNA molecule of the disclosure comprises an expression vector, and the disclosure comprises a method for the introduction of exogenousDNA into the nucleus of cells or tissues of interest with concomitant expression of the exogenous DNA in the cells or tissues of interest.

[0237] In various embodiments, the nucleic acid molecule comprises a DNA. In some embodiments, the DNA is free of an expressible nucleic acid sequence encoding an antigen or antigenic determinant thereof, or a functional variant thereof.

[0238] In some embodiments, the nucleic acid comprising a DNA is encapsulated in an LNP herein.

[0239] In some embodiments, the DNA is a modified DNA. In some embodiments, between 0.1% and 100% of the residues in the modified of the present disclosure are modified. In some embodiments, 0.1% of the residues are modified. In some embodiments, the fraction of modified residues is 0.2%. In some embodiments, the fraction is about 0.3%. In some embodiments, the fraction is about 0.4%. In some embodiments, the fraction is about 0.5%. In some embodiments, the fraction is about 0.6%. In some embodiments, the fraction is about 0.8%. In some embodiments, the fraction is about 1%. In some embodiments, the fraction is about 1.5%. In some embodiments, the fraction is about 2%. In some embodiments, the fraction is about 2.5%. In some embodiments, the fraction is about 3%. In some embodiments, the fraction is about 4%. In some embodiments, the fraction is about 5%. In some embodiments, the fraction is about 6%. In some embodiments, the fraction is about 8%. In some embodiments, the fraction is about 10%. In some embodiments, the fraction is about 12%. In some embodiments, the fraction is about 14%. In some embodiments, the fraction is about 16%. In some embodiments, the fraction is about 18%. In some embodiments, the fraction is about 20%. In some embodiments, the fraction is about 25%. In some embodiments, the fraction is about 30%. In some embodiments, the fraction is about 35%. In some embodiments, the fraction is about 40%. In some embodiments, the fraction is about 45%. In some embodiments, the fraction is about 50%. In some embodiments, the fraction is about 60%. In some embodiments, the fraction is about 70%. In some embodiments, the fraction is about 80%. In some embodiments, the fraction is about 90%. In some embodiments, the fraction is about 100%.

[0240] In some embodiments, the fraction is about less than 5%. In some embodiments, the fraction is about less than 3%. In some embodiments, the fraction is about less than 1%. Insome embodiments, the fraction is about less than 2%. In some embodiments, the fraction is about less than 4%. In some embodiments, the fraction is about less than 6%. In some embodiments, the fraction is about less than 8%. In some embodiments, the fraction is about less than 10%. In some embodiments, the fraction is about less than 12%. In some embodiments, the fraction is about less than 15%. In some embodiments, the fraction is about less than 20%. In some embodiments, the fraction is about less than 30%. In some embodiments, the fraction is about less than 40%. In some embodiments, the fraction is about less than 50%. In some embodiments, the fraction is about less than 60%. In some embodiments, the fraction is about less than 70%.

[0241] In some embodiments, 0.1% of the residues of a given nucleoside (i.e., thymidine, cytidine, guanosine, or adenosine) are modified. In some embodiments, the fraction of the given nucleotide that is modified is 0.2%. In some embodiments, the fraction is about 0.3%. In some embodiments, the fraction is about 0.4%. In some embodiments, the fraction is about 0.5%. In some embodiments, the fraction is about 0.6%. In some embodiments, the fraction is about 0.8%. In some embodiments, the fraction is about 1%. In some embodiments, the fraction is about 1.5%. In some embodiments, the fraction is about 2%. In some embodiments, the fraction is about 2.5%. In some embodiments, the fraction is about 3%. In some embodiments, the fraction is about 4%. In some embodiments, the fraction is about 5%. In some embodiments, the fraction is about 6%. In some embodiments, the fraction is about 8%. In some embodiments, the fraction is about 10%. In some embodiments, the fraction is about 12%. In some embodiments, the fraction is about 14%. In some embodiments, the fraction is about 16%. In some embodiments, the fraction is about 18%. In some embodiments, the fraction is about 20%. In some embodiments, the fraction is about 25%. In some embodiments, the fraction is about 30%. In some embodiments, the fraction is about 35%. In some embodiments, the fraction is about 40%. In some embodiments, the fraction is about 45%. In some embodiments, the fraction is about 50%. In some embodiments, the fraction is about 60%. In some embodiments, the fraction is about 70%. In some embodiments, the fraction is about 80%. In some embodiments, the fraction is about 90%. In some embodiments, the fraction is about 100%.

[0242] In some embodiments, the fraction of the given nucleotide that is modified is less than 8%. In some embodiments, the fraction is about less than 10%. In some embodiments, the fraction is about less than 5%. In some embodiments, the fraction is about less than 3%. In some embodiments, the fraction is about less than 1%. In some embodiments, the fraction is about less than 2%. In some embodiments, the fraction is about less than 4%. In some embodiments, the fraction is about less than 6%. In some embodiments, the fraction is about less than 12%. In some embodiments, the fraction is about less than 15%. In some embodiments, the fraction is about less than 20%. In some embodiments, the fraction is about less than 30%. In some embodiments, the fraction is about less than 40%. In some embodiments, the fraction is about less than 50%. In some embodiments, the fraction is about less than 60%. In some embodiments, the fraction is about less than 70%.

[0243] In some embodiments, the DNA does not activate any pathophysiologic pathways.

[0244] In some embodiments, a DNA comprising an expressible nucleic acid sequence herein is transcribed and translated very efficiently and almost immediately following delivery, and serves as a stable template for mRNA and protein production. In certain instances, a therapeutic agent or antigen encoded by the DNA molecule encapsulated within an LNP herein induces greater production of antigen-specific antibody production as compared to antigen encoded by an mRNA molecule.

[0245] In some embodiments, a nucleic acid molecule herein comprises a nucleic acid sequence encoding a therapeutic agent. In some embodiments, a nucleic acid molecule herein comprises a nucleic acid sequence encoding a plurality of therapeutic agents. In some embodiments, the therapeutic agent is an antigen, antigenic determinant thereof, or a functional variant thereof. In some embodiments, the nucleic acid molecule is an RNA. In some embodiments, the nucleic acid molecule is a DNA. In some embodiments, the nucleic acid molecule comprise one or a plurality of nucleic acid sequences encoding one or more antigens. In one embodiment, the therapeutic agent is a binding molecule (e.g., an antibody or antibody fragment) specific for binding to an antigen. In some embodiments, the RNA or the DNAcomprises one or a plurality of nucleic acid sequences encoding one or more binding molecules.

[0246] In some embodiments, the antigen comprises a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, a tumor- specific antigen, or any combination thereof. In some embodiments, the disclosure includes a nucleic acid molecule encoding an adjuvant.

[0247] In some embodiments, the antigen is encoded by a nucleotide sequence of a nucleic acid molecule. In one embodiment, the antigen is the target of a binding molecule encoded by a nucleotide sequence of a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises RNA, DNA, cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, an RNA-DNA hybrid, LNA, artificial chromosomes or any combination thereof. In certain instances, the nucleic acid sequence comprises one or more additional sequences that encode linker or tag sequences that are linked to an encoded protein, peptide, antigen or antibody by a peptide bond.

[0248] In some embodiments, the composition comprises a nucleic acid sequence which encodes an antigen. For example, In some embodiments, the composition comprises an RNA or DNA molecule comprising a coding sequence encoding an antigen. The antigen may be any molecule or compound, including but not limited to a polypeptide, peptide or protein that induces an adaptive immune response in a subject.

[0249] In some embodiments, the antigen comprises a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the antigen, and therefore the pathogen. In one embodiment, the antigen comprises a fragment of a polypeptide or peptide associated with a pathogen, such that the antigen induces an adaptive immune response against the pathogen.

[0250] In some embodiments, the antigen comprises an amino acid sequence that is substantially homologous to the amino acid sequence of an antigen described herein and retains the immunogenic function of the original amino acid sequence. In some embodiments, the amino acid sequence of the antigen has a degree of identity with respect to the original aminoacid sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%.

[0251] Viral Antigens

[0252] In some embodiments, the antigen comprises a viral antigen, or fragment thereof, or variant thereof. In some embodiments, the viral antigen is from a virus from one of the following families: Adenoviridae, Arenaviridae, Bunyaviridae, Caliciviridae, Coronaviridae, Filoviridae, Hepadnaviridae, Herpesviridae, Orthomyxoviridae, Papovaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In some embodiments, the viral antigen is from papilloma viruses, for example, human papillomoa virus (HPV), human immunodeficiency virus (HIV), polio virus, hepatitis B virus, hepatitis C virus, smallpox virus (Variola major and minor), vaccinia virus, influenza virus, rhinoviruses, dengue fever virus, equine encephalitis viruses, rubella virus, yellow fever virus, Norwalk virus, hepatitis A virus, human T-cell leukemia virus (HTLV-I), hairy cell leukemia virus (HTLV-II), California encephalitis virus, Hanta virus (hemorrhagic fever), rabies virus, Ebola fever virus, Marburg virus, measles virus, mumps virus, respiratory syncytial virus (RSV), herpes simplex 1 (oral herpes), herpes simplex 2 (genital herpes), herpes zoster (varicella-zoster, a.k.a., chickenpox), cytomegalovirus (CMV), for example human CMV, Epstein-Barr virus (EBV), flavivirus, foot and mouth disease virus, chikungunya virus, lassa virus, arenavirus, or cancer causing virus.

[0253] SARS-CoV-2 Antigen

[0254] In one embodiment, the antigen comprises a SARS-CoV-2 antigen or fragment thereof, or variant thereof. The SARS-CoV-2 antigens are those capable of eliciting an adaptive immune response in a mammal against one or more SARS-CoV-2 strain. In some embodiments, the antigen comprises the full length spike protein, a variant thereof, or a fragment thereof. In some embodiments, the fragment of the spike protein comprises the receptor binding domain (RBD).

[0255] In one embodiment, the SARS-CoV-2 antigen contains at least one antigenic epitope that can be effective against particular influenza immunogens against which an immuneresponse can be induced. In some embodiments, the antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact SARS-CoV-2 virus.

[0256] Hepatitis Antigen

[0257] In one embodiment, the antigen comprises a hepatitis virus antigen (i.e., hepatitis antigen), or fragment thereof, or variant thereof. In some embodiments, the hepatitis antigen comprises an antigen or immunogen from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and / or hepatitis E virus (HEV). In some embodiments, the hepatitis antigen is full-length or immunogenic fragments of full-length proteins.

[0258] In one embodiment, the hepatitis antigen comprises an antigen from HAV. For example, In some embodiments, the hepatitis antigen comprises a HAV capsid protein, a HAV non-structural protein, a fragment thereof, a variant thereof, or a combination thereof.

[0259] In one embodiment, the hepatitis antigen comprises an antigen from HCV. For example, In some embodiments, the hepatitis antigen comprises a HCV nucleocapsid protein (i.e., core protein), a HCV envelope protein (e.g., E1 and E2), a HCV non-structural protein (e.g., NS1, NS2, NS3, NS4a, NS4b, NS5a, and NS5b), a fragment thereof, a variant thereof, or a combination thereof.

[0260] In one embodiment, the hepatitis antigen comprises an antigen from HDV. For example, In some embodiments, the hepatitis antigen comprises a HDV delta antigen, fragment thereof, or variant thereof.

[0261] In one embodiment, the hepatitis antigen comprises an antigen from HEV. For example, In some embodiments, the hepatitis antigen comprises a HEV capsid protein, fragment thereof, or variant thereof.

[0262] In one embodiment, the hepatitis antigen comprises an antigen from HBV. For example, In some embodiments, the hepatitis antigen comprises a HBV core protein, a HBV surface protein, a HBV DNA polymerase, a HBV protein encoded by gene X, fragment thereof, variant thereof, or combination thereof. In some embodiments, the hepatitis antigen comprises a HBV genotype A core protein, a HBV genotype B core protein, a HBV genotype C core protein, a HBV genotype D core protein, a HBV genotype E core protein, a HBV genotype Fcore protein, a HBV genotype G core protein, a HBV genotype H core protein, a HBV genotype A surface protein, a HBV genotype B surface protein, a HBV genotype C surface protein, a HBV genotype D surface protein, a HBV genotype E surface protein, a HBV genotype F surface protein, a HBV genotype G surface protein, a HBV genotype H surface protein, fragment thereof, variant thereof, or combination thereof.

[0263] Human Papilloma Virus (HPV) Antigen

[0264] In one embodiment, the antigen comprises a human papilloma virus (HPV) antigen, or fragment thereof, or variant thereof. For example, In some embodiments, the antigen comprises an antigen from HPV types 16, 18, 31, 33, 35, 45, 52, and 58, which cause cervical cancer, rectal cancer, and / or other cancers. In one embodiment, the antigen comprises an antigen from HPV types 6 and 11, which cause genital warts, and are known to be causes of head and neck cancer. For example, In some embodiments, the HPV antigen comprises a HPV E6 or E7 domain, or fragments, or variant thereof from any HPV type.

[0265] RSV Antigen

[0266] In one embodiment, the antigen comprises an RSV antigen or fragment thereof, or variant thereof. For example, In some embodiments, the RSV antigen comprises a human RSV fusion protein (also referred to herein as “RSV F”, “RSV F protein” and “F protein”), or fragment or variant thereof. In one embodiment, the human RSV fusion protein is conserved between RSV subtypes A and B. In some embodiments, the RSV antigen comprises a RSV F protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23994.1). In one embodiment, the RSV antigen comprises a RSV F protein from the RSV A2 strain (GenBank AAB59858.1), or a fragment or variant thereof. In some embodiments, the RSV antigen is a monomer, a dimer or trimer of the RSV F protein, or a fragment or variant thereof. According to the disclosure, In some embodiments, the RSV F protein is in a prefusion form or a postfusion form.

[0267] In one embodiment, the RSV antigen comprises a human RSV attachment glycoprotein (also referred to herein as “RSV G”, “RSV G protein” and “G protein”), or fragment or variant thereof. The human RSV G protein differs between RSV subtypes A and B. In one embodiment, the antigen comprises a RSV G protein, or fragment or variant thereof,from the RSV Long strain (GenBank AAX23993). In one embodiment, the RSV antigen comprises RSV G protein from: the RSV subtype B isolate H5601, the RSV subtype B isolate H1068, the RSV subtype B isolate H5598, the RSV subtype B isolate H1123, or a fragment or variant thereof.

[0268] In other embodiments, the RSV antigen comprises a human RSV non-structural protein 1 (“NS1 protein”), or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV NS1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23987.1). In one embodiment, the RSV antigen comprises RSV non- structural protein 2 (“NS2 protein”), or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1). In one embodiment, the RSV antigen comprises human RSV nucleocapsid (“N”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen is RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989.1). In one embodiment, the RSV antigen comprises human RSV Phosphoprotein (“P”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990.1). In one embodiment, the RSV antigen comprises human RSV Matrix protein (“M”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).

[0269] In still other embodiments, the RSV antigen comprises human RSV small hydrophobic (“SH”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). In one embodiment, the RSV antigen comprises human RSV Matrix protein2-1 (“M2-1”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M2-1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1). In one embodiment, the RSV antigen comprises RSV Matrix protein 2-2 (“M2-2”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV M2-2 protein, or fragment orvariant thereof, from the RSV Long strain (GenBank AAX23997.1). In one embodiment, the RSV antigen comprises RSV Polymerase L (“L”) protein, or fragment or variant thereof. For example, in one embodiment, the RSV antigen comprises RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996.1).

[0270] Influenza Antigen

[0271] In some embodiments, the antigen comprises an influenza antigen or fragment thereof, or variant thereof. The influenza antigens are those capable of eliciting an adaptive immune response in a mammal against one or more influenza serotypes. In some embodiments, the antigen comprises the full-length translation product Hemagglutinin (HA)0, subunit HA1, subunit HA2, a variant thereof, a fragment thereof or a combination thereof. In some embodiments, the influenza hemagglutinin antigen is derived from one or more strains of influenza A serotype H1, influenza A serotype H2, or influenza B.

[0272] In some embodiments, the influenza antigen contains at least one antigenic epitope that can be effective against particular influenza immunogens against which an immune response can be induced. In some embodiments, the antigen may provide an entire repertoire of immunogenic sites and epitopes present in an intact influenza virus.

[0273] In some embodiments, the influenza antigen comprises H1 HA, H2 HA, H3 HA, H5 HA, or a BHA antigen. In some embodiments, the influenza antigen comprises neuraminidase (NA), matrix protein, nucleoprotein, M2 ectodomain-nucleo-protein (M2e-NP), a variant thereof, a fragment thereof, or combinations thereof.

[0274] Human Immunodeficiency Virus (HIV) Antigen

[0275] In some embodiments, the antigen comprises an HIV antigen or fragment thereof, or variant thereof.

[0276] In some embodiments, the HIV antigen comprises an envelope (Env) protein or fragment or variant thereof. For example, In some embodiments, the HIV antigen comprises an Env protein selected from gp120, gp41, or a combination thereof.

[0277] In some embodiments, the HIV antigen comprises at least one of nef, gag, pol, vif, vpr, vpu, tat, rev, or a fragment of variant thereof.

[0278] The HIV antigen may be derived from any strain of HIV. For example, In some embodiments the HIV antigen comprises an antigen from HIV groups M, N, O, and P, and subtype A, HIV subtype B, HIV subtype C, HIV subtype D, subtype E, subtype F, subtype G, subtype H, subtype J, or subtype K.

[0279] Parasite Antigens

[0280] In some embodiments, the antigen comprises a parasite antigen or fragment or variant thereof. In some embodiments, the parasite is a protozoa, helminth, or ectoparasite. In some embodiments, the helminth (i.e., worm) is a flatworm (e.g., flukes and tapeworms), a thorny-headed worm, or a round worm (e.g., pinworms). In some embodiments, the ectoparasite is lice, fleas, ticks, and mites.

[0281] In some embodiments, the parasite is any parasite causing the following diseases: Acanthamoeba keratitis, Amoebiasis, Ascariasis, Babesiosis, Balantidiasis, Baylisascariasis, Chagas disease, Clonorchiasis, Cochliomyia, Cryptosporidiosis, Diphyllobothriasis, Dracunculiasis, Echinococcosis, Elephantiasis, Enterobiasis, Fascioliasis, Fasciolopsiasis, Filariasis, Giardiasis, Gnathostomiasis, Hymenolepiasis, Isosporiasis, Katayama fever, Leishmaniasis, Lyme disease, Malaria, Metagonimiasis, Myiasis, Onchocerciasis, Pediculosis, Scabies, Schistosomiasis, Sleeping sickness, Strongyloidiasis, Taeniasis, Toxocariasis, Toxoplasmosis, Trichinosis, and Trichuriasis.

[0282] In some embodiments, the parasite is Acanthamoeba, Anisakis, Ascaris lumbricoides, Botfly, Balantidium coli, Bedbug, Cestoda (tapeworm), Chiggers, Cochliomyia hominivorax, Entamoeba histolytica, Fasciola hepatica, Giardia lamblia, Hookworm, Leishmania, Linguatula serrata, Liver fluke, Loa loa, Paragonimus - lung fluke, Pinworm, Plasmodium falciparum, Schistosoma, Strongyloides stercoralis, Mite, Tapeworm, Toxoplasma gondii, Trypanosoma, Whipworm, or Wuchereria bancrofti.

[0283] Malaria Antigen

[0284] In some embodiments, the antigen comprises a malaria antigen (i.e., PF antigen or PF immunogen), or fragment thereof, or variant thereof. For example, in one embodiment, the antigen comprises an antigen from a parasite causing malaria. In one embodiment, the malaria causing parasite is Plasmodium falciparum.

[0285] In some embodiments, the malaria antigen comprises one or more of P. falciparum immunogens CS; LSA1; TRAP; CelTOS; and Ama1. The immunogens may be full length or immunogenic fragments of full-length proteins.

[0286] Bacterial Antigens

[0287] In some embodiments, the antigen comprises a bacterial antigen or fragment or variant thereof. In some embodiments, the bacterium is from any one of the following phyla: Acidobacteria, Actinobacteria, Aquificae, Bacteroidetes, Caldiserica, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Elusimicrobia, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Lentisphaerae, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Synergistetes, Tenericutes, Thermodesulfobacteria, Thermotogae, and Verrucomicrobia.

[0288] In some embodiments, the bacterium is a gram-positive bacterium or a gram negative bacterium. In some embodiments, the bacterium is an aerobic bacterium or an anaerobic bacterium. In some embodiments, the bacterium is an autotrophic bacterium or a heterotrophic bacterium. In some embodiments, the bacterium is a mesophile, a neutrophile, an extremophile, an acidophile, an alkaliphile, a thermophile, psychrophile, halophile, or an osmophile.

[0289] In some embodiments, the bacterium is an anthrax bacterium, an antibiotic resistant bacterium, a disease-causing bacterium, a food poisoning bacterium, an infectious bacterium, Salmonella bacterium, Staphylococcus bacterium, Streptococcus bacterium, or tetanus bacterium. In some embodiments, bacterium is a mycobacteria, Clostridium tetani, Yersinia pestis, Bacillus anthracis, methicillin-resistant Staphylococcus aureus (MRSA), or Clostridium difficile.

[0290] Mycobacterium tuberculosis Antigens

[0291] In some embodiments, the antigen comprises a Mycobacterium tuberculosis antigen (i.e., TB antigen or TB immunogen), or fragment thereof, or variant thereof. The TB antigen can be from the Ag85 family of TB antigens, for example, Ag85A and Ag85B. The TB antigen can be from the Esx family of TB antigens, for example, EsxA, EsxB, EsxC, EsxD, EsxE, EsxF, EsxH, EsxO, EsxQ, EsxR, EsxS, EsxT, EsxU, EsxV, and EsxW.

[0292] Fungal Antigens

[0293] In some embodiments, the antigen comprises a fungal antigen or fragment or variant thereof. In some embodiments, the fungus is Aspergillus species, Blastomyces dermatitidis, Candida yeasts (e.g., Candida albicans), Coccidioides, Cryptococcus neoformans, Cryptococcus gattii, dermatophyte, Fusarium species, Histoplasma capsulatum, Mucoromycotina, Pneumocystis jirovecii, Sporothrix schenckii, Exserohilum, or Cladosporium.

[0294] Tumor Antigens

[0295] In some embodiments, the antigen comprises a tumor antigen, including for example a tumor-associated antigen or a tumor-specific antigen. In the context of the present disclosure, “tumor antigen” or “hyperporoliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” refer to antigens that are common to specific hyperproliferative disorders. In certain aspects, the hyperproliferative disorder antigens of the present disclosure are derived from cancers including, but not limited to, primary or metastatic melanoma, mesothelioma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non- Hodgkin's lymphoma, Hodgkins lymphoma, leukemias, uterine cancer, cervical cancer, bladder cancer, kidney cancer and adenocarcinomas such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, and the like.

[0296] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. In one embodiment, the tumor antigen of the present disclosure comprises one or more antigenic cancer epitopes immunogenically recognized by tumor infiltrating lymphocytes (TIL) derived from a cancer tumor of a mammal. The selection of the antigen will depend on the particular type of cancer to be treated or prevented by way of the composition of the disclosure.

[0297] Tumor antigens are well known in the art and include, for example, a glioma- associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M- CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein,PSMA, Her2 / neu, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

[0298] In some embodiments, the tumor antigen comprises one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER- 2 / Neu / ErbB-2. Yet another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor-specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[0299] The type of tumor antigen referred to in the disclosure may also be a tumor- specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA associated antigen is not unique to a tumor cell and instead is also expressed on a normal cell under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells.

[0300] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE,GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor-suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm- 23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO- 029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[0301] Exemplary antigens that can be delivered using the LNPs of the disclosure include, but are not limited to, a SARS-CoV-2 Spike antigen, SARS-CoV-2 Spike protein receptor binding domain (RBD), a hemagglutinin antigen (HA) and the like.

[0302] In some embodiments, the nucleic acid molecule encodes an antigen that induces an adaptive immune response against the antigen. In some embodiments, the therapeutic agent is an antigen that induces an adaptive immune response against the antigen.

[0303] In some embodiments, the RNA or DNA molecule encodes a binding molecule specific for binding to an antigen. In some embodiments, the binding molecule is specific for binding to a protein, peptide, a fragment thereof, or a variant thereof, or a combination thereof from any number of organisms, for example, a virus, a parasite, a bacterium, a fungus, or a mammal. For example, In some embodiments, the binding molecule is specific for binding to an antigen associated with an autoimmune disease, allergy, or asthma. In other embodiments, the binding molecule is specific for binding to an antigen associated with cancer, coronavirus, herpes, influenza, hepatitis B, hepatitis C, human papilloma virus (HPV), ebola, pneumococcus, Haemophilus influenza, meningococcus, dengue, tuberculosis, malaria, norovirus or human immunodeficiency virus (HIV). Exemplary antigens that can be targeted by a binding molecule include, but are not limited to, the antigens described above in detail.

[0304] In some embodiments, the RNA or DNA molecule encodes a biologically active molecule. Exemplary biologically active molecules that can be encoded include, but are not limited to, cytokines, chemokines, costimulatory molecules, and enzymes.

[0305] The nucleotide sequences, as described herein, can comprise sequence variations with respect to a wild-type or parental nucleotide sequence, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a biologically active agent according to the disclosure. Therefore, the scope of the present disclosure includes nucleotide sequences that are substantially homologous to a wild-type or parental nucleotide sequence.

[0306] As used herein, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, advantageously of at least 70%, preferably of at least 85%, and more preferably of at least 95%. A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding an antigen can typically be isolated from a producer organism of the antigen based on the information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

[0307] In some embodiments, the disclosure relates to a construct, comprising a nucleotide sequence encoding an antigen, a binding molecule, a biologically active molecule, or a combination thereof. In some embodiments, the disclosure relates to a construct, comprising a nucleotide sequence encoding an adjuvant.

[0308] Exemplary adjuvants include, but is not limited to, alpha-interferon, gamma- interferon, platelet derived growth factor (PDGF), TNFα, TNFβ, GM-CSF, epidermal growth factor (EGF), cutaneous T cell-attracting chemokine (CTACK), epithelial thymus-expressed chemokine (TECK), mucosae-associated epithelial chemokine (MEC), IL-12, IL-15, MHC,CD80, CD86 including IL-15 having the signal sequence deleted and optionally including the signal peptide from IgE. Other genes which may be useful adjuvants include those encoding: MCP-I, MIP-Ia, MIP-Ip, IL-8, RANTES, L-selectin, P-selectin, E-selectin, CD34, GlyCAM- 1, MadCAM-1, LFA-I, VLA-I, Mac-1, pl50.95, PECAM, ICAM-I, ICAM-2, ICAM-3, CD2, LFA-3, M-CSF, G-CSF, IL-4, mutant forms of IL-18, CD40, CD40L, vascular growth factor, fibroblast growth factor, IL-7, nerve growth factor, vascular endothelial growth factor, Fas, TNF receptor, Fit, Apo-1, p55, WSL-I, DR3, TRAMP, Apo-3, AIR, LARD, NGRF, DR4, DR5, KILLER, TRAIL-R2, TRICK2, DR6, Caspase ICE, Fos, c-jun, Sp-I, Ap-I, Ap-2, p38, p65Rel, MyD88, IRAK, TRAF6, IkB, Inactive NIK, SAP K, SAP-I, JNK, interferon response genes, NFkB, Bax, TRAIL, TRAILrec, TRAILrecDRC5, TRAIL-R3, TRAIL-R4, RANK, RANK LIGAND, Ox40, Ox40 LIGAND, NKG2D, MICA, MICB, NKG2A, NKG2B, NKG2C, NKG2E, NKG2F, TAP 1, TAP2, anti-CTLA4-sc, anti-LAG3-Ig, anti-TIM3-Ig and functional fragments thereof.

[0309] In some embodiments, the construct comprises a first nucleotide sequence encoding an antigen, a binding molecule, or a biologically active molecule, and a second nucleotide sequence encoding an adjuvant.

[0310] In some embodiments, the composition comprises a plurality of constructs, each construct encoding one or more of an antigen, a binding molecule, a biologically active molecule, or a combination thereof. In some embodiments, the composition comprises 1 or more, 2 or more, 5 or more, 10 or more, 15 or more, or 20 or more constructs. In one embodiment, the composition comprises a first construct, comprising a nucleotide sequence encoding an antigen, a binding molecule, a biologically active molecule, or a combination thereof; and a second construct, comprising a nucleotide sequence encoding an adjuvant.

[0311] In some embodiments, the construct is operatively bound to one or more regulatory sequence for the expression of the nucleotide sequence of the disclosure, thus forming an expression cassette. Exemplary regulatory elements include, but are not limited to, a promoter, a leader sequence, a poly-A sequence, enhancers, selection markers, tags and other elements.

[0312] In some embodiments, the composition of the disclosure comprises an RNA or DNA vector or plasmid for expression of an encoded nucleotide sequence. In some embodiments, the composition of the disclosure comprises an RNA or DNA molecule which contains all the elements necessary for expression of an encoded agent (e.g., protein, antigen, antibody, cytokine, chemokine, costimulatory molecule or enzyme) for delivery to the nucleus of a target cell of interest. For example, in one embodiment, the composition comprises an RNA or a DNA expression plasmid encapsulated within a LNP. In certain instances, the LNP enhances cellular and nuclear uptake of the RNA or DNA expression plasmid. In some embodiments, the nucleic acid molecule is an RNA or DNA plasmid comprising a nucleotide sequence encoding a therapeutic agent, an antigen, an antibody, a fragment of antibody or a gene. Thus, in one embodiment, the composition of the present disclosure comprises an RNA or DNA plasmid comprising a nucleotide sequence encoding an antigen. In one embodiment, the composition of the disclosure comprises an RNA or DNA plasmid comprising a nucleotide sequence encoding an antibody, or an antibody fragment. In some embodiments, the composition of the disclosure comprises an RNA or DNA plasmid comprising a nucleotide sequence encoding an adjuvant. In some embodiments, the composition of the disclosure comprises an RNA or DNA plasmid comprising a nucleotide sequence encoding one or more antigens and one or more adjuvants.

[0313] The RNA or DNA can be produced using methods known in the art. RNA or DNA of interest can be from any source. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. The source of the RNA can be, for example, synthetic DNA, cellular RNA, transcripts from genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other DNA, or in vitro transcripts from genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other DNA.

[0314] In one embodiment, a desired expression construct is generated using PCR. In one embodiment, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the DNA is a full length gene of interest of a portion of a gene. The gene caninclude some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In one embodiment, the DNA to be used for PCR is a human gene. In some embodiments, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In some embodiments, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In some embodiments, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

[0315] The nucleic acid sequences coding for the agent of the disclosure can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques.

[0316] Alternatively, a gene of interest can be produced synthetically.

[0317] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, sequencing vectors and vectors optimized for in vitro transcription.

[0318] Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0319] In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of thenucleic acids (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / RNA, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0320] In some embodiments, the nucleic acid molecule, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[0321] In some embodiments, the nucleic acid molecule, when present in the lipid nanoparticles, is formulated in a buffer solution. Exemplary buffers include, but are not limited to, 20% sucrose, SSC and PBS.

[0322] In some embodiments, the composition comprises one or more transfection reagent. In some embodiments, the transfection reagent is a lipid-based transfection reagent. In some embodiments, the transfection reagent is a protein-based transfection reagent. In some embodiments, the transfection reagent is a polyethyleneimine based transfection reagent. In some embodiments, the transfection reagent is calcium phosphate. In some embodiments, the transfection reagent is Lipofectin®, Lipofectamine®, or TransIT®. In some embodiments, the transfection reagent is any other transfection reagent known in the art.

[0323] In some embodiments, the transfection reagent forms a liposome. Liposomes, In some embodiments, increase intracellular stability, increase uptake efficiency and improvebiological activity. In some embodiments, liposomes are hollow spherical vesicles composed of lipids arranged in a similar fashion as those lipids which make up the cell membrane. They have, In some embodiments, an internal aqueous space for entrapping water-soluble compounds and range in size from 0.05 to several microns in diameter. In some embodiments, liposomes can deliver DNA to cells in a biologically active form.

[0324] In some embodiments, the delivery vehicle comprising the RNA or DNA molecules are administered directly to the subject, in vivo. In some embodiments, in vivo delivery of RNA or DNA molecules using the LNP of the disclosure stimulates CD8 immune responses.

[0325] LNP Vaccine

[0326] In one aspect of the disclosure, the compositions described herein are vaccines. For a composition to be useful as a vaccine, the composition must induce an adaptive immune response to the antigen in a cell, tissue, or mammal (e.g., a human). In certain instances, the vaccine induces a protective immune response in the mammal. As used herein, an “immunogenic composition” may comprise an antigen (e.g., a peptide or polypeptide), a nucleic acid encoding an antigen, a cell expressing or presenting an antigen or cellular component, or a combination thereof. In particular embodiments the composition comprises or encodes all or part of any peptide antigen described herein, or an immunogenically functional equivalent thereof. In other embodiments, the composition is in a mixture that comprises an additional immunostimulatory agent or nucleic acids encoding such an agent. Immunostimulatory agents include but are not limited to an additional antigen, an immunomodulator, an antigen presenting cell or an adjuvant. In other embodiments, one or more of the additional agent(s) is covalently bonded to the antigen or an immunostimulatory agent, in any combination. In some embodiments, the antigenic composition is conjugated to or comprises an HLA anchor motif amino acids.

[0327] In the context of the present disclosure, the term “vaccine” refers to a substance that induces immunity upon inoculation into animals.

[0328] A vaccine of the present disclosure may vary in its composition of nucleic acid and / or cellular components. In a non-limiting example, a nucleic acid encoding an antigenmight also be formulated with an adjuvant. In some embodiment, the nucleic acid encoding an antigen is an RNA molecule, optionally encapsulated in an LNP disclosed herein; and the adjuvant is an LNP disclosed herein comprising a DNA molecule free of a nucleic acid encoding an antigen. In some embodiments, the DNA molecule is free of an expressible nucleic acid encoding an antigen. Of course, it will be understood that various compositions described herein may further comprise additional components. For example, one or more vaccine components may be comprised in a lipid, liposome, or lipid nanoparticle. A vaccine of the present disclosure, and its various components, may be prepared and / or administered by any method disclosed herein or as would be known to one of ordinary skill in the art, in light of the present disclosure.

[0329] The induction of the immunity by the expression of the antigen can be detected by observing in vivo or in vitro the response of all or any part of the immune system in the host against the antigen. In some embodiments, DNA-LNP are administered directly to a subject, in vivo.

[0330] For example, a method for detecting the induction of cytotoxic T lymphocytes is well known. A foreign substance that enters the living body is presented to T cells and B cells by the action of APCs. T cells that respond to the antigen presented by APC in an antigen specific manner differentiate into cytotoxic T cells (also referred to as cytotoxic T lymphocytes or CTLs) due to stimulation by the antigen. These antigen stimulated cells then proliferate. This process is referred to herein as “activation” of T cells. Therefore, CTL induction by an epitope of a polypeptide or peptide or combinations thereof can be evaluated by presenting an epitope of a polypeptide or peptide or combinations thereof to a T cell by APC, and detecting the induction of CTL. Furthermore, APCs have the effect of activating B cells, CD4+ T cells, CD8+ T cells, macrophages, eosinophils and NK cells.

[0331] A method for evaluating the inducing action of CTL using dendritic cells (DCs) as APC is well known in the art. DC is a representative APC having a robust CTL inducing action among APCs. In the methods of the disclosure, the epitope of a polypeptide or peptide or combinations thereof is initially expressed by the DC and then this DC is contacted with T cells. Detection of T cells having cytotoxic effects against the cells of interest after the contactwith DC shows that the epitope of a polypeptide or peptide or combinations thereof has an activity of inducing the cytotoxic T cells. Furthermore, the induced immune response can be also examined by measuring IFN-gamma produced and released by CTL in the presence of antigen-presenting cells that carry immobilized peptide or combination of peptides by visualizing using anti-IFN- gamma antibodies, such as an ELISPOT assay.

[0332] Apart from DC, peripheral blood mononuclear cells (PBMCs) may also be used as the APC. The induction of CTL is reported to be enhanced by culturing PBMC in the presence of GM-CSF and IL-4. Similarly, CTL has been shown to be induced by culturing PBMC in the presence of keyhole limpet hemocyanin (KLH) and IL-7.

[0333] The antigens confirmed to possess CTL-inducing activity by these methods are antigens having DC activation effect and subsequent CTL-inducing activity. Furthermore, CTLs that have acquired cytotoxicity due to presentation of the antigen by APC can be also used as vaccines against antigen-associated disorders.

[0334] The induction of immunity by expression of the antigen can be further confirmed by observing the induction of antibody production against the antigen. For example, when antibodies against an antigen are induced in a laboratory animal immunized with the composition encoding the antigen, and when antigen-associated pathology is suppressed by those antibodies, the composition is determined to induce immunity.

[0335] The induction of immunity by expression of the antigen can be further confirmed by observing the induction of CD4+ T cells. CD4+ T cells can also lyse target cells, but mainly supply help in the induction of other types of immune responses, including CTL and antibody generation. The type of CD4+ T cell help can be characterized, as Th1, Th2, Th9, Th17, Tregulatory, or T follicular helper (Tfh) cells. Each subtype of CD4+ T cell supplies help to certain types of immune responses. Of particular interest to this disclosure, the Tfh subtype provides help in the generation of high affinity antibodies.

[0336] Targeting Domain

[0337] In some embodiments, the disclosure comprises targeted delivery of an RNA molecule to a specific cell type of interest with a DNA molecule adjuvant encapsulated in an LNP. In some embodiments, the RNA molecule is a component of an LNP composition of thedisclosure and comprises a targeting domain bound or conjugated to one or more lipid of the LNP. In one embodiment, the targeting domain of the instant disclosure is an antibody, or a fragment thereof, that specifically binds to a surface antigen expressed on a target cell of interest. In some embodiments, the composition comprises one or a plurality of LNPs, wherein the composition is free of an TLR9 agonist or TLR9 ligand or a functional variant thereof, either in solution or anchored in the one or plurality of LNPs. In some embodiments, the composition comprises an RNA molecule encapsulated in a first LNP and comprising a RNA sequence encoding one or a plurality of antigens or antigenic determinants and the composition comprises a second LNP comprising a DNA molecule, wherein the DNA molecule is free of a nucleic acid sequence encoding one or any antigen or antigenic determinant.

[0338] The present disclosure provides, in various embodiments, delivery vehicles comprising antibodies or antibody fragments as targeting domains for targeting the delivery vehicles (e.g., lipid nanoparticles) to a target cell of interest.

[0339] In one embodiment, the targeting domain of the disclosure comprises an antibody, or antibody fragment. In some embodiments, the antibody targeting domain specifically binds to a target of interest. Such antibodies include polyclonal antibodies, monoclonal antibodies, Fab and single chain Fv (scFv) fragments thereof, bispecific antibodies, heteroconjugates, human and humanized antibodies.

[0340] The antibodies may be intact monoclonal or polyclonal antibodies, and immunologically active fragments (e.g., a Fab or (Fab)2 fragment), an antibody heavy chain, an antibody light chain, humanized antibodies, a genetically engineered single chain Fv molecule (Ladner et al, U.S. Pat. No. 4,946,778), or a chimeric antibody, for example, an antibody which contains the binding specificity of a murine antibody, but in which the remaining portions are of human origin. Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.

[0341] Such antibodies may be produced in a variety of ways, including hybridoma cultures, recombinant expression in bacteria or mammalian cell cultures, and recombinant expression in transgenic animals. The choice of manufacturing methodology depends onseveral factors including the antibody structure desired, the importance of carbohydrate moieties on the antibodies, ease of culturing and purification, and cost. Many different antibody structures may be generated using standard expression technology, including full-length antibodies, antibody fragments, such as Fab and Fv fragments, as well as chimeric antibodies comprising components from different species. Antibody fragments of small size, such as Fab and Fv fragments, having no effector functions and limited pharmokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity.

[0342] Peptide targeting moieties

[0343] In one embodiment, the targeting domain of the disclosure comprises a peptide. In some embodiments, the peptide targeting domain specifically binds to a target of interest. In some embodiments, the target of interest is a

[0344] The peptide of the present disclosure may be made using chemical methods. For example, peptides can be synthesized by solid phase techniques (Roberge J Y et al (1995) Science 269: 202-204), cleaved from the resin, and purified by preparative high performance liquid chromatography. Automated synthesis may be achieved, for example, using the ABI 431 A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.

[0345] The peptide may alternatively be made by recombinant means or by cleavage from a longer polypeptide. The composition of a peptide may be confirmed by amino acid analysis or sequencing.

[0346] The variants of the peptides according to the present disclosure may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, (ii) one in which there are one or more modified amino acid residues, e.g., residues that are modified by the attachment of substituent groups, (iii) one in which the peptide is an alternative splice variant of the peptide of the present disclosure, (iv) fragments of the peptides and / or (v) one in which the peptide is fused with another peptide, such as a leader or secretory sequence or a sequence which isemployed for purification (for example, His-tag) or for detection (for example, Sv5 epitope tag). The fragments include peptides generated via proteolytic cleavage (including multi-site proteolysis) of an original sequence. Variants may be post-translationally, or chemically modified. Such variants are deemed to be within the scope of those skilled in the art from the teaching herein.

[0347] Nucleic acid targeting moieties

[0348] In one embodiment, the targeting domain of the disclosure comprises an isolated nucleic acid (e.g., DNA or RNA), including for example, an aptamer, a DNA oligonucleotide or an RNA oligonucleotide. In some embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, in one embodiment, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[0349] The nucleotide sequences of a nucleic acid targeting domain can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting nucleic acid functions as the original and specifically binds to the target of interest.

[0350] Pharmaceutical LNP Compositions

[0351] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0352] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of thedisclosure is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.

[0353] Pharmaceutical compositions that are useful in the methods of the disclosure may be prepared, packaged, or sold in formulations suitable for ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, subcutaneous, intraventricular, intrathecal, intratracheal, intraperitoneal, in utero delivery, or another route of administration or any combination thereof. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

[0354] A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0355] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient.

[0356] In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 10:1 to about 40:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 40:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 20:1. In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of about 10:1.

[0357] In addition to the active ingredient, a pharmaceutical composition of the disclosure may further comprise one or more additional pharmaceutically active agents.

[0358] Controlled- or sustained-release formulations of a pharmaceutical composition of the disclosure may be made using conventional technology.

[0359] As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In some embodiments, parenteral administration is contemplated to include, but is not limited to, intraocular, intravitreal, subcutaneous, intraperitoneal, in utero delivery, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

[0360] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0361] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solutionmay be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0362] A pharmaceutical composition of the disclosure may be prepared, packaged, or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 to about 7 nanometers, and preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder or using a self-propelling solvent / powder- dispensing container such as a device comprising the active ingredient dissolved or suspended in a low-boiling propellant in a sealed container. Preferably, such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nanometers and at least 95% of the particles by number have a diameter less than 7 nanometers. More preferably, at least 95% of the particles by weight have a diameter greater than 1 nanometer and at least 90% of the particles by number have a diameter less than 6 nanometers. Dry powder compositions preferably include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.

[0363] Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50 to 99.9% (w / w) of the composition, and the active ingredient may constitute 0.1 to 20% (w / w) ofthe composition. The propellant may further comprise additional ingredients such as a liquid non-ionic or solid anionic surfactant or a solid diluent (preferably having a particle size of the same order as particles comprising the active ingredient).

[0364] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0365] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0366] As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

[0367] The therapeutic compounds or compositions of the disclosure may be administered prophylactically (i.e., to prevent disease or disorder) or therapeutically (i.e., to treat disease or disorder) to subjects suffering from or at risk of (or susceptible to) developing the disease or disorder. Such subjects may be identified using standard clinical methods. In the context of the present disclosure, prophylactic administration occurs prior to the manifestation of overt clinical symptoms of disease, such that a disease or disorder is prevented or alternatively delayed in its progression. In the context of the field of medicine, the term “prevent” encompasses any activity which reduces the burden of mortality or morbidity from disease. Prevention can occur at primary, secondary and tertiary prevention levels. While primary prevention avoids the development of a disease, secondary and tertiary levels of prevention encompass activities aimed at preventing the progression of a disease and the emergence of symptoms as well as reducing the negative impact of an already established disease by restoring function and reducing disease- related complications.

[0368] Exemplary, non-limiting embodiments of compositions and pharmaceutical compositions herein include the following.

[0369] Some embodiments comprise a composition. In some embodiments, the composition comprises a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof.In some embodiments, the composition further comprises a second LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, one or both of the first LNP or the second LNP are independently any one of the LNPs disclosed herein. See non- limiting examples in the above Lipid Nanoparticles (LNP) section of LNPs that may selected as the first or second LNP.

[0370] Some embodiments comprise a composition. In some embodiments, the composition comprises an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof. In some embodiments, the composition further comprises an LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the LNP is any one of the LNPs disclosed herein. See non- limiting examples in the above Lipid Nanoparticles (LNP) section of LNPs that may selected as the LNP.

[0371] Some embodiments comprise a composition. In some embodiments, the composition comprises an antigenic determinant or functional variant thereof. In some embodiments, the composition further comprises an LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence. In some embodiments, nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the LNP is any one of the LNPs disclosed herein. See non-limiting examples in the above Lipid Nanoparticles (LNP) section of LNPs that may selected as the LNP.

[0372] Some embodiments comprise a vaccine composition. In some embodiments, the composition comprises an antigenic determinant or functional variant thereof or a first nucleicacid molecule comprising a nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the first nucleic acid molecule is a DNA molecule. In some embodiments, the first nucleic acid molecule is an RNA molecule. In some embodiments, the first nucleic acid molecule is encapsulated in a first LNP. In some embodiments, the first nucleic acid molecule is not encapsulated in an LNP. In some embodiments, the composition further comprises a second LNP comprising a second nucleic acid molecule comprising DNA. In some embodiments, second nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence. In some embodiments, the second nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, one or both of the first LNP or the second LNP are independently any one of the LNPs disclosed herein. See non-limiting examples in the above Lipid Nanoparticles (LNP) section of LNPs that may selected as the first or second LNP.

[0373] Some embodiments comprise system comprising a first composition and a second composition. In some embodiments, the first composition comprises an antigenic determinant or functional variant thereof or a first nucleic acid molecule comprising a nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the first nucleic acid molecule is a DNA molecule. In some embodiments, the first nucleic acid molecule is an RNA molecule. In some embodiments, the first nucleic acid molecule is encapsulated in a first LNP. In some embodiments, the first nucleic acid molecule is not encapsulated in an LNP. In some embodiments, the second composition comprises a second LNP comprising a second nucleic acid molecule comprising DNA. In some embodiments, second nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence. In some embodiments, the second nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, one or both of the first LNP or the second LNP are independently any one of the LNPs disclosed herein. See non-limiting examples in the above Lipid Nanoparticles (LNP) section of LNPs that may selected as the first or second LNP. In some embodiments the first composition is contained in a first vessel and the secondcomposition is contained in a second vessel. Some embodiments comprise a kit comprising the first vessel and the second vessel.

[0374] In some embodiments, the nucleic acid molecule comprising DNA of the embodiments herein comprises from about 60% to about 65%, about 70%, about 80%, about 85%, about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 65% to about 70%, about 80%, about 85%, about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 70% to about 80%, about 85%, about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 85%, about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 85% to about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 90% to about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 95 to about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises about 60%, about 65%, about 70%, about 80%, about 85%, about 90%, about 95, or about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA comprises 100% DNA.

[0375] Some embodiments comprise a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an effective amount of a composition or vaccine composition herein and (b) a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises the first composition of a system herein. In some embodiments, the pharmaceutical composition comprises the second composition of a system herein.

[0376] Methods of Delivery

[0377] In one aspect, the present disclosure provides a method for delivery of one or more nucleic acid molecule, therapeutic agent, or any combination thereof.

[0378] In one aspect, the present disclosure provides a method for delivery of a nucleic acid molecule, therapeutic agent, or any combination thereof to a target of interest. Accordingto yet another aspect, a method for in vivo delivery of a nucleic acid molecule, therapeutic agent, or any combination thereof is provided. The method includes in vivo delivery of a DNA molecule.

[0379] In some embodiments, the method includes introducing a DNA molecule into a cell or tissue of a subject in vivo. Examples of such cells or tissues include, but are not limited to, an immune cell, T cell, resident T cells, B cell, natural killer (NK) cell, cancerous cell, cell associated with a disease or disorder, tissue associated with a disease or disorder, brain tissue, central nervous system tissue, pulmonary tissue, apical surface tissue, epithelial cell, endothelial cell, liver tissue, intestine tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscles tissue, joint tissue, tumor cells, diseased tissues, lymph node tissue, lymphatic circulation, or any combination thereof. In various embodiments, the method comprises administering a therapeutically effectively amount of one or more compositions of the present disclosure to a cell or tissue of a subject.

[0380] In some embodiments, the present disclosure provides a method for in vivo delivery of a nucleic acid molecule encoding a therapeutic agent, a biologically active agent, a gene editing agent or any combination thereof. For example, in some embodiments, the present disclosure provides a method for in vivo delivery of a nucleic acid molecule a therapeutic agent, a biologically active agent, a gene editing agent or any combination thereof to a cell or tissue Examples of such cells or tissues include, but are not limited to, an immune cell, T cell, resident T cells, B cell, natural killer (NK) cell, cancerous cell, cell associated with a disease or disorder, tissue associated with a disease or disorder, brain tissue, central nervous system tissue, pulmonary tissue, apical surface tissue, epithelial cell, endothelial cell, liver tissue, intestine tissue, colon tissue, small intestine tissue, large intestine tissue, feces, bone marrow, macrophages, spleen tissue, muscles tissue, joint tissue, tumor cells, diseased tissues, lymph node tissue, lymphatic circulation, or any combination thereof. In various embodiments, the method comprises administering a therapeutically effectively amount of one or more compositions of the present disclosure to a cell or tissue of a subject.

[0381] In one aspect, the method is a gene delivery method. Therefore, in some embodiments, the disclosure encompasses in vivo delivery of DNA molecules comprising genes or gene fragments.

[0382] In one aspect, the method is a gene editing method. Therefore, in some embodiments, the disclosure encompasses in vivo delivery of DNA molecules encoding gene editing molecules (e.g., Cas9, sgRNA or a combination thereof).

[0383] In one embodiment, the method comprises DNA described herein that can be introduced to a target of interest (e.g., cell, tissue, etc.) as a form of transient transfection using the LNP compositions of the present disclosure.

[0384] In one aspect, the present disclosure provides a method for delivery of one or more nucleic acid molecules. In some embodiments, the one or more nucleic acid molecules comprise a composition herein.

[0385] In one embodiment, the method comprises a single administration of the composition. In one embodiment, the method comprises multiple administrations of the composition.

[0386] In some embodiments, the composition is administered by an intradermal delivery route, subcutaneous delivery route, intramuscular delivery route, intraventricular delivery route, intrathecal delivery route, oral delivery route, intravenous delivery route, intratracheal delivery route, intraperitoneal delivery route, in utero delivery route, or any combination thereof.

[0387] In some embodiments, the method for delivery of a nucleic acid molecule, therapeutic agent, or any combination thereof to a target of interest (e.g., cell, tissue, etc.) comprising administering a therapeutically effectively amount of the composition of the present disclosure is concurrently performed with any of a number of different methods, for instance, commercially available methods which include, but are not limited to, cationic liposome mediated transfection using lipofection.

[0388] In certain instances, expressing a protein by delivering the encoding DNA has many benefits over methods that use mRNA. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, which does not provide for long-term expression. Half-lives of mRNA molecules are often short, thus mRNA delivery would need frequent dosing, while DNA provides a template for continuous, long-term mRNA and protein production.

[0389] In order to confirm the presence of the DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Northern blotting and RT-PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunogenic means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.

[0390] In one aspect, the present disclosure also discloses a method for delivery of a nucleic acid molecule, therapeutic agent, or any combination thereof to a subject in need thereof. In various embodiments, the method comprises administering a therapeutically effectively amount of one or more compositions of the present disclosure to the subject. In various embodiments, the method comprises the composition of the present disclosure delivering a nucleic acid molecule, therapeutic agent, or any combination to the subject’s cell, tissue, or both.

[0391] Treatment Methods

[0392] The present disclosure provides methods of inducing an adaptive immune response in a subject comprising administering an effective amount of a composition of the present disclosure. For example, in some embodiments, the composition comprises one or more lipids or LNPs of the present disclosure. In some embodiments, the composition comprises one or more antigens, one or more nucleic acids encoding one or more antigens, or any combination thereof and one or more lipids or LNPs of the present disclosure. In some embodiments, the composition comprises an RNA vaccine, such as a LNP comprising an RNA molecule encoding an antigenic determinant or antigen and an LNP comprising a DNA molecule that is an empty vector (a vector free of an expressible nucleic acid sequence encoding the antigen or antigenic determinant encoded by the RNA molecule).

[0393] In one embodiment, the method provides immunity in the subject to an infection, cancer, or disease or disorder associated with an antigen. The present disclosure thusprovides a method of treating or preventing the infection, cancer, or disease, or disorder associated with the antigen. Exemplary antigens and associated infections, diseases, and tumors are described elsewhere herein.

[0394] For example, the method may be used to treat or prevent a viral infection, bacterial infection, fungal infection, parasitic infection, arthritis, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, fetal disease, genetic disease affecting fetal development, or cancer, depending upon the type of antigen of the administered composition.

[0395] The following are non-limiting examples of cancers that can be treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, childhood; appendix cancer; basal cell carcinoma; bile duct cancer, extrahepatic; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; brain stem glioma, childhood; brain tumor, adult; brain tumor, brain stem glioma, childhood; brain tumor, central nervous system atypical teratoid / rhabdoid tumor, childhood; central nervous system embryonal tumors; cerebellar astrocytoma; cerebral astrocytotna / malignant glioma; craniopharyngioma; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumors of intermediate differentiation; supratentorial primitive neuroectodermal tumors and pineoblastoma; visual pathway and hypothalamic glioma; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; central nervous system lymphoma; cerebellar astrocytoma cerebral astrocytoma / malignant glioma, childhood; cervical cancer; chordoma, childhood; chronic lymphocytic leukemia; chronic myelogenous leukemia; chronic myeloproliferative disorders; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing family of tumors; extragonadal germ cell tumor; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (stomach) cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal tumor (gist); germ cell tumor, extracranial; germ cell tumor, extragonadal; germ cell tumor, ovarian; gestational trophoblastic tumor; glioma;glioma, childhood brain stem; glioma, childhood cerebral astrocytoma; glioma, childhood visual pathway and hypothalamic; hairy cell leukemia; head and neck cancer; hepatocellular (liver) cancer; histiocytosis, langerhans cell; Hodgkin lymphoma; hypopharyngeal cancer; hypothalamic and visual pathway glioma; intraocular melanoma; islet cell tumors; kidney (renal cell) cancer; Langerhans cell histiocytosis; laryngeal cancer; leukemia, acute lymphoblastic; leukemia, acute myeloid; leukemia, chronic lymphocytic; leukemia, chronic myelogenous; leukemia, hairy cell; lip and oral cavity cancer; liver cancer; lung cancer, non- small cell; lung cancer, small cell; lymphoma, aids-related; lymphoma, burkitt; lymphoma, cutaneous T-cell; lymphoma, non- Hodgkin lymphoma; lymphoma, primary central nervous system; macroglobulinemia, Waldenstrom; malignant fibrous histiocvtoma of bone and osteosarcoma; medulloblastoma; melanoma; melanoma, intraocular (eye); Merkel cell carcinoma; mesothelioma; metastatic squamous neck cancer with occult primary; mouth cancer; multiple endocrine neoplasia syndrome, (childhood); multiple myeloma / plasma cell neoplasm; mycosis; fungoides; myelodysplastic syndromes; myelodysplastic / myeloproliferative diseases; myelogenous leukemia, chronic; myeloid leukemia, adult acute; myeloid leukemia, childhood acute; myeloma, multiple; myeloproliferative disorders, chronic; nasal cavity and paranasal sinus cancer; nasopharyngeal cancer; neuroblastoma; non-small cell lung cancer; oral cancer; oral cavity cancer; oropharyngeal cancer; osteosarcoma and malignant fibrous histiocytoma of bone; ovarian cancer; ovarian epithelial cancer; ovarian germ cell tumor; ovarian low malignant potential tumor; pancreatic cancer; pancreatic cancer, islet cell tumors; papillomatosis; parathyroid cancer; penile cancer; pharyngeal cancer; pheochromocytoma; pineal parenchymal tumors of intermediate differentiation; pineoblastoma and supratentorial primitive neuroectodermal tumors; pituitary tumor; plasma celt neoplasm / multiple myeloma; pleuropulmonary blastoma; primary central nervous system lymphoma; prostate cancer; rectal cancer; renal cell (kidney) cancer; renal pelvis and ureter, transitional cell cancer; respiratory tract carcinoma involving the nut gene on chromosome 15; retinoblastoma; rhabdomyosarcoma; salivary gland cancer; sarcoma, ewing family of tumors; sarcoma, Kaposi; sarcoma, soft tissue; sarcoma, uterine; sezary syndrome; skin cancer (nonmelanoma); skin cancer (melanoma); skin carcinoma,Merkel cell; small cell lung cancer; small intestine cancer; soft tissue sarcoma; squamous cell carcinoma, squamous neck cancer with occult primary, metastatic; stomach (gastric) cancer; supratentorial primitive neuroectodermal tumors; T-cell lymphoma, cutaneous; testicular cancer; throat cancer; thymoma and thymic carcinoma; thyroid cancer; transitional cell cancer of the renal pelvis and ureter; trophoblastic tumor, gestational; urethral cancer; uterine cancer, endometrial; uterine sarcoma; vaginal cancer; vulvar cancer; Waldenstrom macroglobulinemia; and Wilms tumor.

[0396] In some embodiments, the composition is administered to a subject having an infection, disease, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, or cancer associated with the antigen. In one embodiment, the composition is administered to a subject at risk for developing the infection, disease, heart disease, cardiovascular disease, neurological disorder or disease, genetic disease, autoimmune disease, or cancer associated with the antigen. For example, the composition may be administered to a subject who is at risk for being in contact with a virus, bacteria, fungus, parasite, or the like. In one embodiment, the composition is administered to a subject who has increased likelihood, though genetic factors, environmental factors, or the like, of developing cancer.

[0397] In some embodiments, the composition is administered by an intradermal delivery route, subcutaneous delivery route, intramuscular delivery route, intraventricular delivery route, intrathecal delivery route, oral delivery route, intravenous delivery route, intratracheal delivery route, intraperitoneal delivery route, in utero delivery route, or any combination thereof.

[0398] In some embodiments, a composition of the present disclosure, comprising a nanoparticle encapsulating an antigen-encoding DNA, induces significantly more adaptive immune response than an unencapsulated DNA molecule with the same sequence. In some embodiments, the composition exhibits an adaptive immune response that is 2-fold greater than its unmodified counterpart. In some embodiments, the adaptive immune response is increased by a 3-fold factor. In some embodiments the adaptive immune response is increased by a 5- fold factor. In some embodiments, the adaptive immune response is increased by a 7-foldfactor. In some embodiments, the adaptive immune response is increased by a 10-fold factor. In some embodiments, the adaptive immune response is increased by a 15-fold factor. In some embodiments the adaptive immune response is increased by a 20-fold factor. In some embodiments, the adaptive immune response is increased by a 50-fold factor. In some embodiments, the adaptive immune response is increased by a 100-fold factor. In some embodiments, the adaptive immune response is increased by a 200-fold factor. In some embodiments, the adaptive immune response is increased by a 500-fold factor. In some embodiments, the adaptive immune response is increased by a 1000-fold factor. In some embodiments, the adaptive immune response is increased by a 2000-fold factor. In some embodiments, the adaptive immune response is increased by another fold difference.

[0399] In some embodiments, “induces significantly more adaptive immune response” refers to a detectable increase in an adaptive immune response. In some embodiments, the term refers to a fold increase in the adaptive immune response (e.g., 1 of the fold increases enumerated above). In some embodiments, the term refers to an increase such that the composition of the present disclosure, comprising a DNA cargo, can be administered at a lower dose or frequency than an isolated DNA molecule with the same species while still inducing an effective adaptive immune response. In some embodiments, the increase is such that the composition of the present disclosure, comprising a DNA cargo, can be administered using a single dose to induce an effective adaptive immune response.

[0400] In some embodiments, a composition of the present disclosure, comprising a DNA cargo, exhibits significantly less innate immunogenicity than an isolated in vitro- synthesized RNA molecule with the same sequence. In some embodiments, the composition of the present disclosure, comprising a DNA cargo, exhibits an innate immune response that is 2- fold less than its isolated counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 5-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicityis reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor. In some embodiments, innate immunogenicity is reduced by another fold difference However, when an RNA molecule is administered with a LNP comprising a DNA molecule that does not encode an antigenic determinant, the RNA molecule comprising a nucleic acid sequence encoding the antigenic determinant induces a stronger immune response in magnitude and specificity against the antigenic determinant than the immune response elicited by the RNA molecule alone.

[0401] In some embodiments, “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In some embodiments, the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above). In some embodiments, the term refers to a decrease such that an effective amount of the composition of the present disclosure, comprising a DNA cargo, can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a decrease such that the composition of the present disclosure, comprising a DNA cargo, can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the recombinant protein. In some embodiments, the decrease is such that the composition of the present disclosure, comprising a DNA cargo, can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the recombinant protein.

[0402] In one aspect, the present disclosure related, in part, to methods of preventing or treating a disease or disorder in a subject in need thereof. In various embodiments, the method comprises administering a therapeutically effectively amount of the composition of the present disclosure to the subject. In some embodiments, the composition delivers a nucleic acid molecule, therapeutic agent, or a combination thereof to a target of interest (e.g., cell, tissue, etc.).

[0403] In some embodiments, the method comprises administering a composition comprising one or more nucleic acid molecules encoding one or more antigens and one or more adjuvant. In one embodiment, the method comprises administering a composition comprising a first nucleic acid molecule encoding one or more antigens and a second nucleic acid molecule encoding one or more adjuvants. In some embodiments, the one or more adjuvants comprise at least one LNP encapsulated nucleic acid molecule comprising DNA. In one embodiment, the method comprises administering a first composition comprising one or more nucleic acid molecules encoding one or more antigens and administering a second composition comprising one or more nucleic acid molecules encoding one or more adjuvants.

[0404] In some embodiments, the method comprises administering to subject a plurality of nucleic acid molecules encoding a plurality of antigens, adjuvants, or a combination thereof. In some embodiments, the plurality of adjuvants comprise at least one LNP encapsulated nucleic acid molecule comprising DNA

[0405] In some embodiments, the method of the disclosure allows for sustained expression of the antigen or adjuvant, described herein, for at least several days following administration. However, the method, in some embodiments, also provides for transient expression, as in some embodiments, the nucleic acid is not integrated into the subject genome.

[0406] In some embodiments, the method comprises administering nanoparticles comprising DNA cargo which provides stable expression of the antigen or adjuvant described herein. In some embodiments, administration of nanoparticles comprising DNA cargo results in little to no innate immune response, while inducing an effective adaptive immune response.

[0407] Administration of the compositions of the disclosure in a method of treatment can be achieved in a number of different ways, using methods known in the art. In one embodiment, the method of the disclosure comprises systemic administration of the subject, including for example enteral or parenteral administration. In some embodiments, the method comprises intradermal delivery of the composition. In some embodiments, the method comprises intravenous delivery of the composition. In some embodiments, the method comprises intramuscular delivery of the composition. In one embodiment, the method comprises subcutaneous delivery of the composition. In one embodiment, the methodcomprises inhalation of the composition. In one embodiment, the method comprises intranasal delivery of the composition. In some embodiments, the methods of treatment are free of a step of inducing TLR9.. In some embodiments, the methods of treatment are free of a step of inducing TLR9 by administration of a TLR9 agonist, ligand or variant thereof.

[0408] It will be appreciated that the composition of the disclosure may be administered to a subject either alone, or in conjunction with another agent.

[0409] The therapeutic and prophylactic methods of the disclosure thus encompass the use of pharmaceutical compositions encoding an antigen, adjuvant, or a combination thereof, described herein to practice the methods of the disclosure. The pharmaceutical compositions useful for practicing the disclosure may be administered to deliver a dose of from ng / kg / day and 100 mg / kg / day. In some embodiments, the disclosure envisions administration of a dose which results in a concentration of the compound of the present disclosure from 10 nM to 10 µM in a mammal. In some embodiments, the disclosure envisions administration of a dose which results in a concentration of the compound of the present disclosure from 10nM to 10 mM in a mammal. In some embodiments, the disclosure envisions administration of a dose which results in a concentration of the compound of the present disclosure from about 10 nM to about 10 mM in a mammal. In some embodiments, the disclosure envisions administration of a dose which results in a concentration of the compound of the present disclosure from about 10 nM to about 100 nM, about 1 µM, about 10 µam, about 100µ, about 1mM, or about 10 mM in a mammal. In some embodiments, the disclosure envisions administration of a dose which results in a concentration of the compound of the present disclosure of about 10 nM, about 100 nM, about 1 µM, about 10 µam, about 100µ, about 1mM, or about 10 mM in a mammal.

[0410] Typically, dosages which may be administered in a method of the disclosure to a mammal, preferably a human, range in amount from 0.01 μg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. Preferably, the dosage of the compound will vary from about 0.1 μg to about 10 mg per kilogram of bodyweight of the mammal. More preferably, the dosage will vary from about 1 μg to about 1 mg per kilogram of body weight of the mammal.

[0411] The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

[0412] In some embodiments, administration of the composition or vaccine of the present disclosure may be performed by single administration or boosted by multiple administrations.

[0413] In one embodiment, the disclosure includes a method comprising administering one or more compositions encoding one or more antigens or adjuvants described herein. In some embodiments, the method has an additive effect, wherein the overall effect of the administering the combination is approximately equal to the sum of the effects of administering each antigen or adjuvant. In other embodiments, the method has a synergistic effect, wherein the overall effect of administering the combination is greater than the sum of the effects of administering each antigen or adjuvant.

[0414] In one embodiment, the method comprises the systemic administration of the composition into the subject, including for example intradermal administration. In some embodiments, the method comprises administering a plurality of doses to the subject. In some embodiments, the method comprises administering a single dose of the composition, where the single dose is effective in inducing an adaptive immune response.

[0415] In some embodiments, the disclosure relates to a method of enhancing an immune response in a subject in need thereof. In some embodiments, the method comprises administering to the subject a composition comprising a first lipid nanoparticle (LNP) and a nucleic acid molecule comprising DNA. In some embodiments, the LNP comprises SM-102 or a derivative thereof. In some embodiments, the LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the LNP comprises polyethylene glycol (PEG). In some embodiments, the LNP comprises (i) SM-102 or a derivative thereof, (ii) a cholesterol molecule or a derivative thereof, (iii) 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or a derivative thereof, and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the LNP is chosen from any LNP herein.

[0416] In some embodiments, (ii), (iii), (iv) and (v) form a lipid nanoparticle encapsulating the nucleic acid molecule. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof. In some embodiments, the method further comprises administering a second lipid nanoparticle comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or a functional variant thereof. In some embodiments, the antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease. In some embodiments the antigenic determinant or a functional variant thereof is chosen from any antigen or immune target herein.

[0417] In some embodiments, the composition further comprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.1 micrograms to about 400 micrograms. In some embodiments, the composition further comprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms. In some embodiments, the lipid to DNA weight ratio of the first lipid nanoparticle is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1. In some embodiments, the nucleic acid molecule comprising DNA comprises one or a plurality of: a cDNA molecule, a linear DNA molecule, a circular plasmid DNA molecule, mini-circle DNA molecule, a rolling circle amplified DNA product, an artificial chromosome, a replicating DNA or any combination thereof. In some embodiments, the first lipid nanoparticle comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension.

[0418] In some embodiments, the first lipid nanoparticle comprises a spheroid shape and comprises a diameter from about 50 to about 180 nanometers at its longest width dimension.

[0419] In some embodiments, the method comprises administering a population of first lipid nanoparticles encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules. In some embodiments, the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 3 nucleic acid molecules.

[0420] In some embodiments, the nucleic acid molecule comprising DNA is a DNA plasmid. In some embodiments, the first lipid nanoparticle has a zeta potential of from about - 2 millivolts to about -22 millivolts. In some embodiments, the composition comprises a plurality of first lipid nanoparticles; and wherein the first lipid nanoparticles have an average zeta potential of from about -0.5 mV to about -6 mV. In some embodiments, the composition comprises a plurality of first lipid nanoparticles. In some embodiments, the first lipid nanoparticles have an average diameter of from about 70 to about 77 nanometers.

[0421] In some embodiments, the nucleic acid molecule comprising DNA is free of RNA. In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding one or a plurality of antigenic determinants or functional variants thereof.

[0422] In some embodiments, the immune response is against an antigenic determinant or functional variant thereof. In some embodiment, enhancing an immune response comprises enhancing a immune response to a vaccine. In some embodiment, enhancing an immune response comprises enhancing an antigen-specific immune response in a subject in need thereof before, after or simultaneous administration of a vaccine comprising an antigenic determinant or functional variant thereof or a nucleic acid sequence encoding an antigenic determinant or functional variant thereof.

[0423] In some embodiments, the disclosure relates to a method of vaccinating a subject in need thereof. In some embodiments, the method comprises administering acomposition to the subject, wherein the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof and at least a first LNP. In some embodiments, the at least first LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the at least first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the at least first LNP in any LNP herein.

[0424] In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA.

[0425] In some embodiments, the method of vaccinating comprises administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises an RNA molecule encoding an antigenic determinant or functional variant thereof. The antigenic determinant or functional variant thereof may be for an antigen or immune target herein.

[0426] In some embodiments, the second composition comprises at least a first LNP. In some embodiments, the at least a first LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the at least first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and(iv) polyethylene glycol (PEG). In some embodiments the at least first LNP is any LNP herein. In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, the at least first LNP comprises (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the at least first LNP comprises nucleic acid molecule comprising DNA. In some embodiments, the nucleic acid molecule comprises from about 80% to about 100% DNA. In some embodiments, the at least first LNP comprises PEG is DMG- PEG-2000 or a derivative thereof. In some embodiments, the method further comprises a second lipid nanoparticle encapsulating the RNA molecule. In some embodiments, the second LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the second LNP. In some embodiments, the at least first LNP comprises antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease. In some embodiments, the at least first LNP composition further comprises a plurality of LNPS comprising DNA with a total mass amount of from about 0.1 micrograms to about 400 micrograms of DNA In some embodiments, the at least first LNP comprises composition further comprises one or a plurality of LNPs comprising nucleic acid molecules comprising DNA with a total mass amount of from about 0.002 micrograms to about 4 micrograms of DNA. In some embodiments, the at least first LNP comprises lipid to DNA weight ratio of the LNP is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1. In some embodiments, the at least first LNP comprises nucleic acid molecule comprising DNA is a DNA plasmid. In some embodiments, the at least first LNP comprises at least first LNP comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension. In some embodiments, the at least first LNP comprises composition comprises a population of first LNPs encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first LNPs are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules. In some embodiments, the at least first LNP comprises LNP comprising a nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a variant thereof. In some embodiments, the at least first LNP comprises LNP comprising anucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding any antigenic determinant or a variant thereof.

[0427] In some embodiments, the disclosure relates to a composition comprising a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; and a second LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, the first LNP and the second LNP independently comprise any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may independently comprise the first LNP or the second LNP. In some embodiments, the second LNP comprises SM-102 or a derivative thereof. In some embodiments, the second LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the second LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the second LNP comprises polyethylene glycol (PEG). In some embodiments, the second LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the LNP is an LNP herein. In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof.. In some embodiments, the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of any nucleic acid sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, the first LNP comprises SM-102 or a derivative thereof. In some embodiments, the first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first LNP comprises polyethylene glycol (PEG). In some embodiments, the first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50:about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof.

[0428] In some embodiments, the disclosure relates to a pharmaceutical composition comprising (a) an effective amount of a composition and (b) a pharmaceutically acceptable carrier. In some embodiments, the composition comprises a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; and a second LNP comprising a nucleic acid molecule comprising DNA. In some embodiments, the first LNP and the second LNP independently comprise any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may independently comprise the first LNP or the second LNP. In some embodiments, the second LNP comprises SM-102 or a derivative thereof. In some embodiments, the second LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the second LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the second LNP comprises polyethylene glycol (PEG). In some embodiments, the second LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the second LNP is any LNP herein. In some embodiments, the nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of any nucleic acid sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, the first LNP comprises SM-102 or a derivative thereof. In some embodiments, the first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first LNP comprises polyethylene glycol (PEG). In some embodiments, the first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterolmolecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the first LNP is any LNP herein. In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof.

[0429] In some embodiments, the disclosure relates to a pharmaceutical composition comprising: (a) a first LNP comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; (b) a second LNP comprising a DNA molecule comprising a nucleic acid sequence free of any antigenic determinant or functional variant thereof; and (c) a pharmaceutically acceptable carrier. In some embodiments, the first LNP and the second LNP independently comprise any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may independently comprise the first LNP or the second LNP. In some embodiments, the first and second LNPs comprise SM-102 or a derivative thereof. In some embodiments, the first and second LNPs comprise a cholesterol molecule or a derivative thereof. In some embodiments, the first and second LNPs comprise 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first and second LNPs comprise polyethylene glycol (PEG). In some embodiments, the first and second LNPs comprise: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, the first and second LNPs are independently selected from any LNP herein. In some embodiments, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the RNA sequence encodes an antigenic determinant. In some embodiments, the antigenic determinant is from any antigen or immune target herein. In some embodiments, the RNA sequence encodes a viral antigenic determinant or an antigenic determinant associated with hyperproliferative disease.

[0430] In some embodiments, the disclosure relates to a method of vaccinating a subject in need thereof . In some embodiments, the method comprises (a) administering to the subject a composition comprising a vaccine and a vaccine adjuvant; or (b) administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises a vaccine and the second composition comprises a vaccine adjuvant.

[0431] In some embodiments, the vaccine comprises an antigenic determinant or a functional variant thereof or comprises a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof. In some embodiments, the antigenic determinant or a functional variant thereof is from any antigen or immune target herein. In some embodiments, the vaccine adjuvant comprises at least a first LNP. In some embodiments, the at least a first LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, the at least first LNP is any LNP herein. In some embodiments, the vaccine comprises an RNA molecule comprising an RNA sequence that encodes the antigenic determinant or functional variant thereof. In some embodiments, the vaccine comprises a lipid nanoparticle comprising from about 1 to about 20 RNA molecules. In some embodiments, the nucleic acid molecule comprising DNA is free of RNA. In some embodiments, the nucleic acid molecule comprising DNA is free of a DNA sequence encoding any antigenic determinant or functional variant thereof. In some embodiments, the nucleic acid molecule comprising DNA is free of an expressible DNA sequence encoding any antigenic determinant or functional variant thereof. In someembodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

[0432] In some embodiments, the disclosure relates to a method of preventing or treating a pathogen infection in a subject in need thereof. In some embodiments, the method comprises (a) administering a composition to the subject. In some embodiments, the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof from the pathogen and at least a first LNP. In some embodiments, the at least a first LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In some embodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, the at least first LNP is any LNP herein.

[0433] In some embodiments, the method of preventing or treating a pathogen infection comprises (b) administering a first composition and a second composition to the subject. In some embodiments, the first composition comprises an RNA molecule encoding an antigenic determinant or functional variant thereof. In some embodiments, the second composition comprises at least a first LNP. In some embodiments, the at least a first LNP comprises any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the at least a first LNP. In some embodiments, the at least first LNP comprises SM-102 or a derivative thereof. In some embodiments, the at least first LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, the at least first LNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the at least first LNP comprises polyethylene glycol (PEG). In someembodiments, the at least first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG). In some embodiments, (i), (ii), (iii) and (iv) form the first LNP and the LNP encapsulates a nucleic acid molecule comprising DNA. In some embodiments, (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA. In some embodiments, the nucleic acid molecule comprising DNA is free of RNA. In some embodiments, the PEG is DMG-PEG-2000 or a derivative thereof. In some embodiments, the at least first LNP is any LNP herein.

[0434] In some embodiments, the method of preventing or treating a pathogen infection further comprises a second lipid nanoparticle encapsulating the RNA molecule. The second LNP may be any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non- limiting LNPs that may comprise the second LNP. In some embodiments, the method of preventing or treating a pathogen infection, the DNA molecule is free of a nucleic acid sequence encoding the antigenic determinant or functional variant thereof. In some embodiments, the method of preventing or treating a pathogen infection, the DNA molecule is free of an expresssible nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

[0435] In some embodiments, the disclosure relates to a method of enhancing an antigen-specific immune response in a subject in need thereof. In some embodiments, the method comprises (a) administering a vaccine comprising an antigenic determinant or functional variant thereof or administering a vaccine comprising a nucleic acid sequence encoding the antigenic determinant or functional variant thereof; and (b) administering an LNP comprising a DNA molecule is free of a nucleic acid sequence encoding the antigenic determinant or functional variant thereof. The LNP may be any LNP herein. See the Lipid Nanoparticles (LNP) section for exemplary, non-limiting LNPs that may comprise the LNP. In some embodiments, the LNP comprises SM-102 or a derivative thereof. In some embodiments, the LNP comprises a cholesterol molecule or a derivative thereof. In some embodiments, theLNP comprises 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the LNP comprises polyethylene glycol (PEG). In some embodiments, the LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG In some embodiments, (i), (ii), (iii) and (iv) form the LNP and the LNP encapsulates the DNA molecule. In some embodiments, the at least first LNP is any LNP herein. In some embodiments, the DNA molecule is free of a nucleic acid sequence encoding any antigenic determinant or functional variant thereof.

[0436] In some embodiments, the disclosure relates to a method of treating or preventing an infection or a disorder in a subject in need thereof. In some embodiments, the method comprising administering to the subject a pharmaceutical composition herein. In some embodiments, if the method treats or prevents an infection, the antigenic determinant or functional variant thereof is from a pathogen. In some embodiments, if the method treats or prevents a disorder, the antigenic determinant or functional variant thereof is associated with the disorder. In some embodiments, the disorder is a hyperproliferative disorder and the antigenic determinant or functional variant thereof is from a cell associated with a hyperproliferative disorder.

[0437] In some embodiments, the disclosure relates to a cell comprising any composition(s) or any pharmaceutical composition(s) herein. EXAMPLES

[0438] Embodiments are further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the disclosure should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0439] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilizethe present disclosure and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure. Example 1: Lipid nanoparticle delivery of plasmid DNA elicits robust adaptive immunity

[0440] In this study a platform is described for formulating plasmid DNA within ionizable LNPs for in vivo delivery as a vaccine. Humoral and cellular immune responses were compared from DNA-LNP formulations encoding two antigens in comparison with nucleoside- modified mRNA-LNP or adjuvanted p...

Claims

CLAIMS 1. A method of enhancing an immune response in a subject in need thereof comprising: (i) administering to the subject a first composition comprising a first lipid nanoparticle (LNP) and a nucleic acid molecule comprising DNA.

2. The method of claim 1, wherein the method further comprises administering an antigen or antigenic determinant thereof or a functional variant thereof to the subject.

3. The method of claim 2, wherein the step of administering an antigen or antigenic determinant thereof to the subject comprises administering a second composition comprising a second lipid nanoparticle and an RNA molecule comprising an RNA sequence encoding the antigenic determinant or a functional variant thereof.

4. The method of claim 3, wherein the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigen or antigenic determinant thereof or functional variant thereof.

5. The method of claim 1 or 2, wherein the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding an antigen or antigenic determinant thereof or functional variant thereof.

6. The method of any one of claims 1 through 5, wherein the LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG).

7. The method of claim 6, wherein (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

8. The method of claim 6 or claim 7, wherein (ii), (iii), (iv) and (v) form a lipid nanoparticle encapsulating the nucleic acid molecule.

9. The method of any one of claims 6 through 8, wherein the PEG is DMG-PEG-2000 or a derivative thereof.

10. The method of any one of claims 1 through 5, wherein the LNP comprises: ALC-0315, ALC-0159, DSPC, and cholesterol.

11. The method of any one of claims 1 through 5, wherein the LNP comprises: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

12. The method of claim 11, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

13. The method of claim 11 or claim 12, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms,and at least one of R3and R4comprises at least two sites of unsaturation.

14. The method of claim 11 or claim 12 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24alkyl, substituted C12-C24alkenyl, substituted C12-C24 alkynyl, or substituted C12-C24 acyl; R3and R4are independently substituted C1-C6 alkyl, substituted C1-C6 alkenyl, or substituted C1-C5 alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

15. The method of any one of claims 11 through 14, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

16. The method of any one of claims 11 through 14, wherein the phospholipid compriseslecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

17. The method of any one of claims 11 through 16, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

18. The method of any one of claims 1 through 5, wherein the LNP comprises: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula X,Formula XIV.

19. The method of any one of claims 1 through 18, wherein the method further comprises administering a second lipid nanoparticle comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or a functional variant thereof.

20. The method of any one of claims 1 through 19, wherein the antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease.

21. The method of any one of claims 1 through 20, wherein the composition furthercomprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.1 micrograms to about 400 micrograms.

22. The method of any one of claims 1 through 20, wherein the composition further comprises one or a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms.

23. The method of any one of claims 1 through 22, wherein the lipid to DNA weight ratio of the first lipid nanoparticle is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1.

24. The method of any one of claims 1 through 23, wherein the nucleic acid molecule comprising DNA comprises one or a plurality of: a cDNA molecule, a linear DNA molecule, a circular plasmid DNA molecule, mini-circle DNA molecule, a rolling circle amplified DNA product, an artificial chromosome, a replicating DNA or any combination thereof.

25. The method of any one of claims 1 through 24, wherein the first lipid nanoparticle comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension.

26. The method of any one of claims 1 through 25, wherein the first lipid nanoparticle comprises a spheroid shape and comprises a diameter from about 50 to about 180 nanometers at its longest width dimension.

27. The method of claim 26 further comprising a population of first lipid nanoparticles encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules.

28. The method of claim 27, wherein the population of first lipid nanoparticles are homogenous in shape and comprise from about 1 to about 3 nucleic acid molecules 29. The method of any claims 1 through 28, wherein the nucleic acid molecule comprising DNA is a DNA plasmid.

30. The method of any one of claims 1 through 29, wherein the first lipid nanoparticle has a zeta potential of from about -2 millivolts to about -22 millivolts.

31. The method of any one of claims 1 through 30, wherein the composition comprises a plurality of first lipid nanoparticles; and wherein the first lipid nanoparticles have an average zeta potential of from about -0.5 mV to about -6 mV.

32. The method of any one of claims 1 through 31, wherein the composition comprises a plurality of first lipid nanoparticles; and wherein the first lipid nanoparticles have an average diameter of from about 70 to about 77 nanometers.

33. The method of any one of claims 1 through 32 wherein the nucleic acid molecule comprising DNA is free of RNA.

34. The method of any one of claims 1 through 33, wherein the immune response is against an antigenic determinant or a functional variant thereof.

35. A method of vaccinating a subject in need thereof comprising: (a) administering a composition to the subject, wherein the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof and at least a first LNP, wherein the LNP encapsulates a nucleic acid molecule comprising DNA; or (b) administering a first composition and a second composition to the subject; wherein the first composition comprises an RNA molecule encoding an antigenicdeterminant or functional variant thereof; and the second composition comprises at least a first LNP, wherein the at least first LNP encapsulates a nucleic acid molecule comprising DNA.

36. The method of claim 35, wherein the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

37. The method of claim 35 or 36, wherein the LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG), and wherein (i), (ii), (iii) and (iv) form the first LNP.

38. The method of claim 37, wherein (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

39. The method of claim 37 or 38, wherein the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA.

40. The method of any one of claims 37 through 39, wherein the PEG is DMG-PEG-2000 or a derivative thereof.

41. The method of any one of claims 35 through 40, wherein the RNA molecule is encapsulated in a second LNP.

42. The method of any one of claims 35, 36, or 41, wherein one or more of the LNP, the first LNP, or the second LNP comprise: ALC-0315, ALC-0159, DSPC, and cholesterol.

43. The method of any one of claims 35, 36, or 41, wherein one or more of the LNP, the first LNP, or the second LNP comprise: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

44. The method of claim 43, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

45. The method of claim 43 or claim 44, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

46. The method of claim 43 or claim 44 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII,wherein R1and R2are independently substituted C12-C24 alkyl, substituted C12-C24 alkenyl, substituted C12-C24 alkynyl, or substituted C12-C24 acyl; R3and R4are independently substituted C1-C6alkyl, substituted C1-C6alkenyl, or substituted C1-C5alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

47. The method of any one of claims 43 through 46, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

48. The method of any one of claims 43 through 46, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

49. The method of any one of claims 43 through 48, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

50. The method of any one of claims 35, 36, or 41, wherein one or more of the LNP, the first LNP, or the second LNP comprise: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula XII,Formula XIV.

51. The method of any one of claims 35 through 50, wherein the antigenic determinant is a viral antigen or an antigenic determinant associated with a hyperproliferative disease.

52. The method of any one of claims 35 through 51, wherein the composition further comprises a plurality of LNPS comprising DNA with a total mass amount of from about 0.1 micrograms to about 400 micrograms of DNA.

53. The method of any one of claims 35 through 52, wherein the composition further comprises one or a plurality of LNPs comprising nucleic acid molecules comprising DNA with a total mass amount of from about 0.002 micrograms to about 4 micrograms of DNA.

54. The method of any one of claims 35 through 53, wherein the lipid to DNA weight ratio of the LNP is from about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1.

55. The method of any one of claims 35 through 54, wherein the nucleic acid molecule comprising DNA is a DNA plasmid.

56. The method of any one of claims 35 through 55, wherein the at least first LNP comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension.

57. The method of any one of claims 35 through 56 wherein the composition comprises a population of first LNPs encapsulating one or a plurality of nucleic acid molecules comprising DNA, wherein the population of first LNPs are homogenous in shape and comprise from about 1 to about 10 nucleic acid molecules.

58. The method of any one of claims 53 through 56, wherein the LNP comprising a nucleic acid molecule comprising DNA is free of a nucleic acid sequence encoding any antigenic determinant or a variant thereof.

59. A composition comprising a first lipid nanoparticle (LNP) comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; and a second LNP comprising a nucleic acid molecule comprising DNA.

60. The composition of claim 59, wherein the nucleic acid molecule comprising DNA is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

61. The composition of claim 59, wherein the nucleic acid molecule comprising DNA is free of any expressible nucleic acid sequence encoding any antigenic determinant or functional variant thereof.

62. The composition of any one of claims 59 through 61, wherein the second LNP comprises: (i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG).

63. The composition of any one of claims 59 through 62, wherein the first LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG).

64. The composition of claim 62 or 63, wherein, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

42.

65. The composition of any one of claims 62 through 64, wherein the PEG is DMG- PEG-2000 or a derivative thereof.

66. The composition of any one of claims 59 through 61, wherein the LNP comprises: ALC-0315, ALC-0159, DSPC, and cholesterol.

67. The composition of any one of claims 59 through 61, wherein the LNP comprises (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

68. The composition of claim 67, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

69. The composition of claim 67 or claim 68, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

70. The composition of claim 67 or claim 68 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24 alkyl, substituted C12-C24 alkenyl, substituted C12-C24alkynyl, or substituted C12-C24acyl; R3and R4are independently substituted C1-C6alkyl, substituted C1-C6alkenyl, or substituted C1-C5alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

71. The composition of any one of claims 67 through 70, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

72. The composition of any one of claims 67 through 70, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

73. The composition of any one of claims 67 through 70, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG- modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG- modified dialkylglycerol, or a mixtures of two or more thereof.

74. The composition of any one of claims 59 through 61, wherein the LNP comprises: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula XII,Formula XIV.

75. The composition of any one of claims 59 through 74, wherein the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA.

76. A pharmaceutical composition comprising (a) an effective amount of the composition of any one of claims 59 through 75; and (b) a pharmaceutically acceptable carrier.

77. A pharmaceutical composition comprising: (a) a first LNP comprising an RNA molecule comprising an RNA sequence encoding an antigenic determinant or functional variant thereof; (b) a second LNP comprising a DNA molecule free of any expressible nucleic acid sequence encoding any antigenic determinant or functional variant thereof; and (c) a pharmaceutically acceptable carrier 78. The pharmaceutical composition of claim 77, wherein one or both of the first and second LNPs comprise: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG); and, wherein, if (i), (ii), (iii) and (iv) are present, they are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

79. The pharmaceutical composition of claim 77, wherein one or both of the first and second LNPs comprise: ALC-0315, ALC-0159, DSPC, and cholesterol.

80. The pharmaceutical composition of claim 77, one or both of the first and second LNPs comprise: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

81. The pharmaceutical composition of claim 80, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

82. The pharmaceutical composition of claim 80 or claim 81, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

83. The pharmaceutical composition of claim 80 or claim 81 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24 alkyl, substituted C12-C24 alkenyl, substituted C12-C24alkynyl, or substituted C12-C24acyl; R3and R4are independently substituted C1-C6alkyl, substituted C1-C6alkenyl, or substituted C1-C5alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen fromnitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

84. The pharmaceutical composition of any one of claims 80 through 83, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

85. The pharmaceutical composition of any one of claims 80 through 83, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl- phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, ordilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

86. The pharmaceutical composition of any one of claims 80 through 85, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

87. The pharmaceutical composition of claim 77, one or both of the first and second LNPs comprise: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula XII,Formula XIV.

88. The pharmaceutical composition of any one of claims 77 through 87, wherein RNA sequence encodes a viral antigenic determinant or an antigenic determinant associated with hyperproliferative disease.

89. A method of vaccinating a subject in need thereof comprising: (a) administering to the subject a composition comprising a vaccine and a vaccine adjuvant; or (b) administering a first composition and a second composition to the subject; wherein the first composition comprises a vaccine and the second composition comprises a vaccine adjuvant; wherein the vaccine comprises an antigenic determinant or a functional variant thereof or comprises a nucleic acid sequence encoding the antigenic determinant or a functional variant thereof; wherein the vaccine adjuvant comprises at least a first LNP, wherein the at least first LNP; and wherein the first LNP encapsulates a nucleic acid molecule comprising a DNA.

90. The method of claim 89, wherein the first LNP comprises:(i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG); wherein (i), (ii), (iii) and (iv) form the first LNP encapsulates a nucleic acid molecule comprising DNA.

91. The method of claim 90, wherein (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

92. The method of claim 89, wherein the first LNP comprises: ALC-0315, ALC-0159, DSPC, and cholesterol.

93. The method of claim 89, wherein the first LNP comprises: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

94. The method of claim 93, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

95. The method of claim 93 or claim 94, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3 alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

96. The method of claim 93 or claim 94 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24alkyl, substituted C12-C24alkenyl, substituted C12-C24alkynyl, or substituted C12-C24acyl; R3and R4are independently substituted C1-C6 alkyl, substituted C1-C6 alkenyl, or substituted C1-C5 alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

97. The method of any one of claims 93 through 96, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or withoutunsaturation.

98. The method of any one of claims 93 through 96, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

99. The method of any one of claims 93 through 98, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

100. The method of claim 89, wherein the first LNP comprises: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula IX,Formula XIII, orFormula XIV.

101. The method of any one of claims 89 through 100, wherein the vaccine comprises an RNA molecule comprising an RNA sequence that encodes the antigenic determinant or functional variant thereof.

102. The method of claim 101, wherein the vaccine comprises a lipid nanoparticlecomprising from about 1 to about 20 RNA molecules.

103. The method of any one of claims 89 through 102, wherein the nucleic acid molecule comprising DNA is free of RNA.

104. The method of any one of claims 89 through 103, wherein the nucleic acid molecule comprising DNA is free of a DNA sequence encoding any antigenic determinant or functional variant thereof.

105. A method of preventing or treating a pathogen infection in a subject in need thereof comprising: (a) administering a composition to the subject, wherein the composition comprises: an RNA molecule encoding an antigenic determinant or functional variant thereof from the pathogen and at least a first LNP, wherein the at least first LNP comprises, wherein the first LNP encapsulates a nucleic acid molecule comprising DNA; or (b) administering a first composition and a second composition to the subject; wherein the first composition comprises an RNA molecule encoding an antigenic determinant or functional variant thereof; and the second composition comprises at least a first LNP, wherein the first LNP encapsulates a nucleic acid molecule comprising DNA.

106. The method of claim 105, wherein the method further comprises a second LNP encapsulating the RNA molecule.

107. The method of claim 105 or 106, wherein the DNA molecule is free of a nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

108. The method of any one of claims 105 through 107, wherein one or both of the first LNP or the second LNP comprise: (i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG); wherein (i), (ii), (iii) and (iv) form one or both of the first LNP or the second LNP.

109. The method of claim 108, wherein (i), (ii), (iii) and (iv) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

110. The method of claim 108 or 109, wherein the PEG is DMG-PEG-2000 or a derivative thereof.

111. The method of any of claims 6 through 8, wherein the PEG is DMG-PEG-2000 or a derivative thereof.

112. The method of any one of claims 105 through 107, wherein one or both of the first LNP or the second LNP comprise: ALC-0315, ALC-0159, DSPC, and cholesterol.

113. The method of any one of claims 105 through 107, wherein one or both of the first LNP or the second LNP comprise: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

114. The method of claim 113, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the total lipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

115. The method of claim 113 or claim 114, wherein cationic and / or ionizable lipidcomprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

116. The method of claim 113 or claim 114 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24alkyl, substituted C12-C24alkenyl, substituted C12-C24 alkynyl, or substituted C12-C24 acyl; R3and R4are independently substituted C1-C6alkyl, substituted C1-C6alkenyl, or substituted C1-C5alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

117. The method of any one of claims 113 through 116, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

118. The method of any one of claims 113 through 116, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

119. The method of any one of claims 113 through 118, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

120. The method of any one of claims 105 through 107, wherein one or both of the first LNP or the second LNP comprise: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula XII,Formula XIV.

121. The method of any one of claims 105 through 120, wherein the nucleic acid molecule comprising DNA comprises from about 80% to about 100% DNA.

122. A method of enhancing an antigen-specific immune response in a subject in need thereof comprising: (a) administering a vaccine comprising an antigenic determinant or functional variant thereof or administering a vaccine comprising a nucleic acid sequence encoding the antigenic determinant or functional variant thereof; and (b) administering an LNP comprising a DNA molecule, wherein the DNA molecule is free of an expressible nucleic acid sequence encoding the antigenic determinant or functional variant thereof.

123. The method of claim 58, wherein the LNP comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; (iii) 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and (iv) polyethylene glycol (PEG); wherein (i), (ii), (iii) and (iv) form the LNP and the LNP encapsulates the DNA molecule.

124. The method of claim 122, wherein the LNP comprises: ALC-0315, ALC-0159, DSPC, and cholesterol.

125. The method of claim 122, wherein the LNP comprises: (a) a cationic and / or ionizable lipid, (b) a phospholipid, (c) a structural lipid, and (d) a PEGylated lipid.

126. The method of claim 125, wherein (a) the cationic and / or ionizable lipid comprises from about 40 mol % to about 60 mol % of the total lipid present in the LNP, (b) the phospholipid comprises from about 5 mol % to about 20 mol % of the total lipid present in the LNP, (c) the structural lipid comprises from about 30 mol % to about 50 mol % of the totallipid present in the LNP, and (d) the PEGylated lipid comprising from about 0.05 mol % to less than 0.5 mol % of the total lipid present in the LNP.

127. The method of claim 125 or claim 126, wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula V or Formula VI,Formula V Formula VI, wherein R1and R2are independently selected and are H or C1-C3alkyls, R3and R4are independently selected and are alkyl groups having from about 10 to about 20 carbon atoms, and at least one of R3and R4comprises at least two sites of unsaturation.

128. The method of claim 125 or claim 126 wherein cationic and / or ionizable lipid comprises a cationic lipid of Formula VII,Formula VII, wherein R1and R2are independently substituted C12-C24 alkyl, substituted C12-C24 alkenyl, substituted C12-C24 alkynyl, or substituted C12-C24 acyl; R3and R4are independently substituted C1-C6alkyl, substituted C1-C6alkenyl, or substituted C1-C5alkynyl or R3and R4may join to form a substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen; R5is either absent or hydrogen or C1-C6 alkyl to provide a quaternary amine; m, n, and p are independently 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and Y and Z are independently O, S, or NH.

129. The method of any one of claims 125 through 128, wherein the phospholipid comprises a lipid of Formula VIII,Formula VIII, wherein R and R′ are independently selected from fatty acid moieties with or without unsaturation.

130. The method of any one of claims 125 through 128, wherein the phospholipid comprises lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleyol-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1- carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl- phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl- phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, or dilinoleoylphosphatidylcholine, and mixtures of two or more thereof.

131. The method of any one of claims 125 through 130, wherein the PEG lipid comprises a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modifiedceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerols, or a PEG-modified dialkylglycerol, or a mixtures of two or more thereof.

132. The method of claim 122, wherein the LNP comprises: a lipid having the structure of Formula IX, Formula X, Formula XI, Formula XII, Formula XIII, or Formula XIV,Formula XII,Formula XIII, orFormula XIV.

134. A method of treating or preventing an infection or a disorder in a subject in need thereof comprising administering to the subject the pharmaceutical composition of any one of claims 76 through 88; wherein, if the method treats or prevents an infection, the antigenic determinant or functional variant thereof is from a pathogen; and wherein, if the method treats or prevents a disorder, the antigenic determinant or functional variant thereof is associated with the disorder.

135. The method of claim 134, wherein the disorder is a hyperproliferative disorder and the antigenic determinant or functional variant thereof is from a cell associated with a hyperproliferative disorder.

136. A cell comprising any of the compositions of claims 59 through 75 or the pharmaceutical compositions of any one of claims 76 through 88.

Images