Lipid nanoparticles and methods of use for DNA delivery

Abstract

The present disclosure relates to lipids and compositions thereof. In various aspects of the invention, the compositions are lipid nanoparticle compositions used to deliver various DNA molecules and / or therapeutic agents to selected targets, such as cells for gene delivery, and / or to prevent or treat diseases or disorders in a subject in need thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 601,398, filed November 21, 2023, entitled “Lipid Nanoparticles and Methods of Use for DNA Delivery;” U.S. Provisional Application No. 63 / 550,819, filed February 7, 2024 and entitled “Lipid Nanoparticles and Methods of Use for DNA Delivery;” and U.S. Provisional Application No. 63 / 707,732, filed October 15, 2024 and entitled Lipid Nanoparticles and Methods of Use for DNA Delivery;” all of which are incorporated herein by reference in their entireties. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention 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 invention. SEQUENCE LISTING

[0003] The electronic sequence listing filed herewith with the filename “WIST-014-PCT_SL,” created November 18, 2024, and having a file size of 48,276 bytes is incorporated herein by reference 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 Modema (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 in vitro single cell 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 (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). Critically, LNP-encapsulated DNA could be of value as it retains the positive product profile of DNA vaccines while obviates the need for device delivery. SUMMARY

[0010] In some embodiments, the invention relates to a composition for in vivo delivery of a DNA molecule to a cell, the composition comprising a lipid nanoparticle (LNP) comprising or encapsulating a DNA molecule.

[0011] 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.

[0012] In some embodiments, the LNP comprises a combination of SM-102 or derivative thereof, cholesterol or a derivative thereof, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof and DMG-PEG-2000 or a derivative thereof at a molar ratio of 50 (SM-102) to 38.5 (Cholesterol) to 10 (DSPC) to 1.5 (DMG-PEG-2000), or 50:38.5:10:1.5. In some embodiments, the LNP comprises a combination of SM-102 or derivative thereof, cholesterol or a derivative thereof, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof and DMG-PEG-2000 or a derivative thereof at a molar ratio of 50 (SM-102 derivative) to 38.5 (Cholesterol) to 10 (DSPC) to 1.5 (DMG-PEG-2000), or about 50: about 38.5: about 10: about 1.5.

[0013] 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 Ri is an alkyl or heteroalkyl group comprising from about 7 to about 25 carbon atoms. In some embodiments, Ri comprises 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 Ri comprises two non-contiguous alkyl or heteroalkyl groups covalently bound to a COO group. In some embodiments n is 1 or 2, and Ri comprises a first and a second noncontiguous 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 Ri is a C12 to C28 monoglyceride, alkenyl, alkyl, aryl, or aralkyl.

[0014] In some embodiments, the invention relates to compositions comprising a lipid nanoparticle encapsulating a DNA cargo molecule. In some embodiments, the DNA cargo molecule is encapsulated within the lipid nanoparticle with an efficiency of at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least 97%, at least 98%, at least 99%, or greater than 99%. In some embodiments, the DNA cargo molecule is encapsulated within the lipid nanoparticle with an efficiency of about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than 99%. In some embodiments, the DNA cargo molecule is encapsulated within the lipid nanoparticle with an efficiency of from about 85% to about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93% to about 94%, about 95%, about 96%, about 97%, about 98%, about 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 pg / mL with lipid containing ethanol at a volumetric ratio of 1:3 (ethanol: citrate buffer) using microfluidic mixing device.

[0015] In some embodiments, the DNA molecule comprises cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, artificial chromosomes, replicating DNA, cosmid or any combination thereof.

[0016] In some embodiments, the DNA molecule comprises a nucleotide sequence encoding a therapeutic agent.

[0017] In some embodiments, the DNA molecule comprises a nucleotide sequence encoding an antigenic molecule. In some embodiments, the antigenic molecule comprises a viral antigen, or a fragment thereof. In some embodiments, the viral antigen is an influenza HA antigen or SARS-CoV-2 antigen. In some embodiments, the viral antigen comprises SEQ ID NO:2 or SEQ ID NO:4, or a functional variant thereof comprising about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

[0018] In some embodiments, the DNA molecule comprises a nucleotide sequence encoding a co-stimulatory molecule, a cytokine or a chemokine.

[0019] In some embodiments, the DNA molecule comprises a nucleotide sequence encoding an antibody or a fragment thereof.

[0020] In some embodiments, the delivery vehicle further comprises a targeting domain to target the LNP to a specific cell of interest. In some embodiments, the targeting domain comprises a binding molecule specific for binding to a surface antigen expresses on the surface of the cell of interest.

[0021] In some embodiments, the DNA molecule comprises cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, artificial chromosomes, replicating DNA or any combination thereof. In some embodiments, the DNA molecule comprises a nucleotide sequence encoding a therapeutic agent. In some embodiments, the DNA molecule encodes one or more antigens. In some embodiments, the antigen is a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, or a tumor-specific antigen.

[0022] In some embodiments, the disclosure relates to a method of preventing or treating a disease or disorder in a subject in need thereof, the method comprising administering a therapeutically effectively amount of at least one composition for in vivo delivery of a DNA molecule to a cell, the delivery, the composition comprising a lipid nanoparticle (LNP) comprising or encapsulating a DNA molecule to the subject. In some embodiments, the LNP comprises a combination of SM-102, cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and DMG-PEG-2000 at a molar ratio of 50 (SM-102) to 38.5 (Cholesterol) to 10 (DSPC) to 1.5 (DMG-PEG-2000).

[0023] In some embodiments, the composition is useful for a method of delivery of a DNA molecule encoding a therapeutic agent to the nucleus of a target cell of interest. In some embodiments, the disclosure relates to a method of treating or preventing infection, disease or a disorder, wherein the disease or disorder is a viral infection. In some embodiments, the viral infection is influenza infection or SARS-CoV-2 infection. In some embodiments, the composition delivers a DNA molecule encoding a therapeutic agent to the nucleus of a target cell of interest. In some embodiments, the disease or disorder is a viral infection. In some embodiments, the viral infection is influenza infection or SARS-CoV-2 infection.

[0024] In some embodiments, the disclosure relates to a method of in vivo delivery of DNA molecules, the method comprising administering to the subject a therapeutically effectively amount of a composition, the composition comprising a lipid nanoparticle (LNP) comprising or encapsulating a DNA molecule. In some embodiments, the LNP comprises a combination of SM-102, cholesterol, 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC) and DMG-PEG-2000 at a molar ratio of about 50 (SM-102) to about 38.5 (Cholesterol) to about 10 (DSPC) to about 1.5 (DMG-PEG-2000). In some embodiments, the DNA molecule comprises cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, artificial chromosomes, replicating DNA or any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings particular embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0026] FIG. 1A through FIG. IC depict the ratio of lipid to DNA formulation impacts immunogenicity. FIG. 1A depicts representative FACS plots of Tfh cells from mice immunized with 2 pg 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 CD 19- CD4+. FIG. IB 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. IC) 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.

[0027] FIG. 2A through FIG. 2C depict data demonstrating that the influenza virus HA-expressing DNA-LNP induces robust GC responses. FIG. 2A depicts representative FACS plots of Tfh cells from mice immunized with 2 pg 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.

[0028] FIG. 3A through FIG. 3H depict data demonstrating that Influenza HA DNA-LNP induces serum responses comparable to mRNA-LNP and adjuvanted protein and is thermostable. FIG. 3 A depicts the area under the curve (AUC) of biweekly serum ELISA data. FIG. 3B depicts the serum endpoint titers at week 8 to A / Califomia / 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.

[0029] FIGS. 4A through FIG. 41 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. Pregated on live CD3+ CD4- CD8+ CD44+ CD62L-. (FIG. 4B) CD107a+ CD8+ T cells shown as in (FIG. 4A). (FIG. 4C) TNFa+ 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) TNFa+ CD4+ T cells shown as in (FIG. 4D). (FIG. 4F) IL-2+ CD4+ T cells shown as in (FIG. 4D). FIG. 4G - FIG. 41 depict data demonstrating the frequency of activated CD8+ T cells expressing IFNy (FIG. 4G), CD 107a (FIG. 4H), or TNFa (FIG. 41) 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. 41, 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.

[0030] FIG. 5A through FIG. 5H depict data demonstrating that SARS-CoV-2 spikeexpressing DNA-LNP elicits robust GC and serum antibody responses. (FIG. 5A) Representative FACS plots of Tfh cells from mice immunized with 2 pg 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 CD 19-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), D614Gfull 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 to compare 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.

[0031] FIG. 6A through 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) TNFa+ 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) TNFa+ 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.

[0032] 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.

[0033] 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 2pg of HA DNA-LNP and serum titers were followed longitudinally, reported as raw OD (FIG. 8A) or endpoint titer calculated against naive animals (FIG. 8B). Dots represent individual animals; n=15 animals per group. Plots show geometric mean with geometric SD.

[0034] FIG. 9A through 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), TNFa (FIG. 9B), or IL-2 (FIG. 9C) 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.

[0035] FIG. 10A through 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. IOC) 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.

[0036] FIGS. 11A through 11J illustrate initial immune characterization of HA DNA-LNP formulations and immunogenicity. FIG. 11 A: Representative FACS plots of GC B cells from mice immunized with 2 pg 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 CD 19+ 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. 1 ID: Frequency of activated Tfh cells expressed as a percentage of CD44+ CD4+ T cells. Pre-gated on live CD19- CD4+. FIG. 1 IE: IFNy ELISpot of splenocytes 14 days post immunization. FIGS. 11F and 11G: Fold change induction of cytokines in DLNs at 4 hours (FIG. 1 IF) and 24 hours (FIG. 11G) post immunization quantified using Luminex. FIGS. 11H and 111: ELISpot assay measuring IFNa (FIG. 11H) and ZENy (FIG. Ill) 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 111); data pooled from two independent experiments (FIGS. 11 A-l IE, 11H, and 111) or from one independent experiment (FIGS. HF and 11G). Plots show geometric mean with geometric SD. Unpaired one-way ANOVA adjusted 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.

[0037] FIGS. 12A through 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 2pg HA DNA-LNP, HA mRNALNP, or 1 pg 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), CDllb+ mDCs (FIG. 12G), CD103+ mDCs (h), pDCs (FIG. 121), 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.

[0038] FIGS. 13A through 13G illustrated single cell transcriptomics elucidates proactivation 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 naive, mRNA-LNP, and protein in adjuvant immunization in NK cells (FIG. 13C), neutrophils (FIG. 13D), monocytes (FIG. 13E), pDCs (FIG. 13F), and eDCs (FIG. 13G). Data represent one independent experiment of 10 pooled mouse popliteal LNs per group.

[0039] FIGS. 14A through 14N illustrate influenza virus HA-expressing DNA-LNP induces robust GC and serum responses. FIG. 14A: Representative FACS plots of Tfh cells from mice immunized with 2 pg of pVAX DNA-LNP, CA09 HA DNA-LNP, or CA09 HA mRNA-LNP. (b-c) Bar plots show quantification of frequency (FIG. 14B) and numbers (FIG. 14C) 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 CD 19- CD4+. FIG. 14D: Representative FACS plots of total GC B cells, (e-f) Bar plots show quantification of frequency (FIG. 14E) and numbers (FIG. 14F). Frequency is expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ (FIG. 14G) Representative FACS plots of CA09 HA-specific GC B cells. FIGS. 14H and 141: Bar plots show frequency (FIG. 14H) and numbers (FIG. 141). 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. Referring to the previous FIGS. 3B and 3D-3G: Serum endpoint titers at week 8 to various H1N1 Has, and FIG. 3C AUC of serum binding antibodies to A / Guangdong-Maonan / SWL1536 / 2019 (FIG. 14K), and A / Victoria / 4897 / 2022 (FIG. 14L). FIGS. 150 and 15P: HAI titers to A / Netherlands / 602 / 2009 (FIG. 14M) and A / New York City / PV63249 / 2022 (FIG. 14N). Dots represent individual animals; n=9-10 animals per group; data pooled from two independent experiments. Plots show geometric mean with geometric SD. Unpaired oneway 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, **** pCO.OOOl.

[0040] FIGS. 15A through 151 illustrate SARS-CoV-2 spike-expressing DNA-LNP elicits serum antibody responses and is protective in lethal challenge. FIG. 15A: Schematic of immunization and challenge scheme. Mice were immunized once with 2 pg 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. 15B-15E: Serum endpoint titers at Week 8 post immunization to wild-type spike RBD (FIG. 15B), D614G full length spike (FIG. 15C), B.1.617.2 (Delta) full length spike (FIG. 15D), and BA.2 full length spike (FIG. 15E). FIG. 15F: Serum neutralization ID50 against wild-type SARS-CoV-2 pseudovirus. FIGS. 15G and 15H: Weight loss (FIG. 15G) and survival (FIG. 15H) of mice challenged with 1x105 PFU mouse-adapted SARS-CoV-2 with an 80% weight loss cutoff. (FIG. 151) 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.

[0041] FIGS. 16A through 161 illustrate modulation of lipid to DNA ratio does not affect T cell responses. FIGS. 16A-16C: Biophysical characterization of DNA-LNPs at different N / P ratios. (FIG. 16A) Particle size (FIG. 16B) Poly dispersity index (PDI) (FIG. 16C) Zeta potential. FIG. 16D-16I: Cytokine / marker expressing effector CD8+ and CD4+ T cells; CD107a+ CD8+ T cells (FIG. 16D), IFNy+ CD8+ T cells (FIG. 16E), TNFa+ CD8+ T cells (FIG. 16F), IFNy+ CD4+ T cells (FIG. 16G), TNFa+ CD4+ T cells (FIG. 16H), IL-2+ CD4+ T cells (FIG. 161). Cells pre-gated on live CD3+ CD4- CD8+ CD44+ CD62L- (FIGS. 16D-16F) or live CD4+ CD8- CD44+ CD62L- (FIGS. 16G-16I). Dots represent individual animals; n=8-9 animals per group (FIGS. 16D-16I). 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.

[0042] FIGS. 17A through 17D illustrate Activation signature of CD4+ T cells and IFNy+ NK cells 24 hours post immunization. FIG. 17A: Representative FACS plots of CD69+ CD4+ T cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3+ NKp46-. FIG. 17B: Bar plot showing quantification of CD69+ CD4+ T cells as in (FIG. 17A) in the popliteal DLN, iliac DLN, and spleen. FIG. 17C: Representative FACS plots of IFNv+ NK cells 24 hours post immunization in the popliteal DLN. Pregated on live CD3- NKp46 +. FIG. 17D: Bar plot showing quantification of IFNy+ NK cells as in (FIG. 17C) 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. 17B and 17D). * p<0.05, ** p<0.01, *** p<0.001, ”” p<0.0001. Related to FIGS. 2A-2C.

[0043] FIGS. 18A through 181 illustrate Innate immune population frequency and activation signature in the spleen 24 hours post immunization. FIGS. 18A-18H: Frequency of total rDCs (FIG. 18A), CD1 lb+ rDCs (FIG. 18B), CD8a+ rDCs (FIG. 18C), total mDCs (FIG. 18D), CDllb+ mDCs (FIG. 18E), CD103+ mDCs (FIG. 18F), neutrophils (FIG. 18G), and pDCs (FIG. 18H) in the spleen expressed as a frequency of CD45+ cells. FIG. 181: Quantification of CD86 expression on all innate populations in FIGS. 18A-18H. 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.

[0044] FIGS. 19A through 19E illustrate overlapping genes with active vaccination groups demonstrates IFN-associated gene signature. Upregulated and downregulated genes among DNA-LNP, mRNA-LNP, and protein in adjuvant immunized mice relative to Naive. FIG. 19A: NK cells. FIG. 19B: Neutrophils. FIG. 19C: Monocytes. FIG. 19D: pDC. FIG. 19E: cDC.

[0045] FIG. 20 illustrates T and NK cell activation gating strategy. Gating strategy for activation of CD4+ T, CD8+ T, and NK cells.

[0046] FIGS. 21A and 21B illustrate innate immune population gating strategy. FIGS. 21A and 21B: Gating strategies for innate immune populations in the DLN (FIG. 21A) and spleen (FIG. 2IB).

[0047] FIGS. 22A and 22B illustrate germinal center and T cell gating strategy. FIGS. 22A and 22B: Gating strategies for germinal center Tfh and GC B cells (FIG. 22A) and T cell responses in the spleen (FIG. 22B).

[0048] FIG. 23 illustrates myeloid population flow cytometry - Lymph node and spleen suspensions were resuspended in Fixable Viability Dye eF780 (eBioscience) in FACS buffer for 10 min at RT. Cells were next washed before resuspension with a surface stain cocktail for 30 min at RT containing the following antibodies: BUV395 anti-mouse Ly-6G (Clone 1A8, BD), BUV563 anti-mouse CD8a (Clone 53-6.7, BD), BUV805 anti-mouse B220 (Clone RA3-6B2, BD), BV421 anti-mouse CD86 (Clone GL-1, Biolegend), BV605 antimouse CDllb (Clone Ml / 70, BD), BV650 anti-mouse CD19 (Clone 1D3, Biolegend), BV785 anti-mouse Ly-6C (Clone HK1.4, Biolegend), FITC anti-mouse TCRP (Clone H57-597, Biolegend), PerCP-Cy5.5 anti-mouse CD11c (Clone N418, Biolegend), PE anti-mouse PDCA-1 (Clone 927, Biolegend), PE-Cy7 anti-mouse CD 103 (Clone 2E7, Biolegend), AF647 antimouse I-A / I-E (Clone M5 / 114.15.2, BD), and AF700 anti-mouse CD45 (Clone 30-F11, Biolegend). Cells were washed and resuspended in FACS buffer and acquired as above.

[0049] FIG. 24 illustrates myeloid population flow cytometry - Lymph node and spleen suspensions were resuspended in Fixable Viability Dye eF780 (eBioscience) in FACS buffer for 10 min at RT. Cells were next washed before resuspension with a surface stain cocktail for 30 min at RT containing the following antibodies: BUV395 anti-mouse Ly-6G (Clone 1A8, BD), BUV563 anti-mouse CD8a (Clone 53-6.7, BD), BUV805 anti-mouse B220 (Clone RA3-6B2, BD), BV421 anti-mouse CD86 (Clone GL-1, Biolegend), BV605 antimouse CD1 lb (Clone Ml / 70, BD), BV650 anti-mouse CD19 (Clone 1D3, Biolegend), BV785 anti-mouse Ly-6C (Clone HK1.4, Biolegend), FITC anti-mouse TCRP (Clone H57-597, Biolegend), PerCP-Cy5.5 anti-mouse CD11c (Clone N418, Biolegend), PE anti-mouse PDCA-1 (Clone 927, Biolegend), PE-Cy7 anti-mouse CD103 (Clone 2E7, Biolegend), AF647 antimouse I-A / I-E (Clone M5 / 114.15.2, BD), and AF700 anti-mouse CD45 (Clone 30-F11, Biolegend). Cells were washed and resuspended in FACS buffer and acquired as above.

[0050] FIG. 25 illustrates a study design.

[0051] FIGS. 26A and 26B illustrate IFNv ELISpot Week 2 Post Boost (FIG. 26A) and HA-specific IgG in Serum (FIG. 26B) under the study design of FIG. 25.

[0052] FIGS. 27A and 27B illustrate Wk 4 endpoint (FIG. 27A) and Wk 2 post boost endpoint (FIG. 27B) under the study design of FIG. 25.

[0053] FIG. 28 illustrates the foot and mouth disease virus and the genomic structure thereof.

[0054] FIG. 29 illustrates a schematic representation of FMDV-Asl-Shamir89 DNA vaccine constructs.

[0055] FIG. 30 illustrates FMDV-As-l-Shamier 89 DNA gel analysis and amino acid sequence.

[0056] FIG. 31 illustrates a schematic representation of FMDV-A24cruzeiro constructs and includes SEQ ID NO: 8.

[0057] FIG. 32 illustrates an amino acid sequence comparison between Shamier and Cruzeiro VP4, VP2, and 2A. Figure discloses SEQ ID NOS 9—14, respectively, in order of appearance.

[0058] FIG. 33 illustrates an amino acid sequence comparison between Shamier and Cruzeiro VP3 and VP1. Figure discloses SEQ ID NOS 15-18, respectively, in order of appearance.

[0059] FIGS. 34A through 34D illustrate cellular immune responses elicited by FMDV-A24cruzeiro vaccines. The y-axis represents TFN-ySFU / (106 cells).

[0060] FIGS. 35A through 35D illustrate cellular immune responses elicited by FMDV-Asl-Shamir89 vaccines. The y-axis represents ZFN-ySFU / (106 cells).

[0061] FIGS. 36A and 36B illustrate induction of antibodies in mice following FMDV DNA vaccine administration.

[0062] FIGS. 37A and 37B illustrate Abs binding (Total IgG) from an FMDV -Immune Reagent Study.

[0063] FIG. 38 illustrates poxviral diversity versus protection.

[0064] FIGS. 39A and 39B compare DNA multi-antigen vaccine to DryVax.

[0065] FIG. 40 illustrate monkey pox antigens.

[0066] FIG. 41 compares SynCon™ vaccines versus conventional consensus vaccines.

[0067] FIGS. 42A and 42B illustrate percent survival in naive versus vaccinated ferret (FIG. 42A) and maximum weight loss in survivors (FIG. 42B).

[0068] FIGS. 43A and 43B illustrate physiology related to Growth hormone-releasing hormone (GHRH).

[0069] FIGS. 44A through 44G illustrate HA DNA-LNP induces potent memory responses in mice. FIG. 44 A) Schematic of immunization regimen. Mice were immunized once with 2 pg of pVAX DNA-LNP, HA DNA-LNP, HA mRNA-LNP, or 1 pg adjuvanted HA protein and populations ere profiled at 378 days post prime. FIG. 44B IFNy secreting cells in splenocytes by ELISpot. FIG. 44C fFNy-secreting effector CD8 T cells by flow cytometry. Pre-gated on live CD3+ CD4' CD8+ CD44+ CD62L'. FIG. 44D CA09 HA-specific ASC responses in bone marrow by ELISpot. FIG. 44E) Representative FACS plot of CA09 HA-specific MBCs. FIG. 44F and 44G Bar plots show frequency FIG. 44F and numbers FIG. 44G). Frequency expressed as a percentage of IgD' IgM' memory B cells. Pre-gated on live dump' CD19+ CD38+ GL7' IgD' IgM'. Dots represent individual animals; n=9-10 animals per group. Plots show mean with SD FIG. 44B) or geometric mean with geometric SD FIGS. 44C, 44D, 44F, and 44G. Unpaired one-way ANOVA or two-way ANOVA FIG. 44B adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. * p<0.05, ** p<0.01, *** p<0.001,     p<0.0001.

[0070] FIGS. 45A through 45G illustrate HA DNA-LNP induces potent memory responses in rabbits. FIG. 45A Schematic of immunization regimen. New Zealand white rabbits were immunized twice on Day 0 and Day 28 with 50 pg of either naked HA DNA, HA DNA-LNP, or HA mRNA-LNP and immune profiling was performed longitudinally. FIGS. 45B-45D IFNy ELISpot on PBMCs at Day 42 FIG. 45B, Day 105 FIG. 45C, and Day 202 FIG. 45D. FIG. 45E AUC of total A / California / 04 / 2009 HA-specific serum IgG ELISA data. FIG. 45F and 45G HAI titers to A / Netherlands / 602 / 2009 FIG. 45F and A / New York City / PV63249 / 2022 FIG. 45G). Dots represent individual animals; n=5 animals per group. Plots show mean with SD FIGS. 45B-45D or geometric mean with geometric SD FIGS. 45E-45G. Unpaired one-way ANOVA or two-way ANOVA FIGS. 45B-45D adjusted for multiple comparisons with Bonferroni corrections was used to compare groups. ANOVA performed at the final timepoint for FIGS. 45E-45G. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

[0071] FIGS. 46A through 46E illustrate SARS-CoV-2 JN. 1 spike DNA-LNP induces specific binding and neutralizing antibody responses. FIG. 46A Layout of immunization scheme. ACE2-Tg mice were vaccinated twice separated by 3 weeks. Mice were primed with wild-type BNT162b2 (0.5ug) and boosted with either the wild-type / BA.4 / 5 bivalent BNT162b2 vaccine (5ug) or JN. 1 spike DNA-LNP (5ug). Negative control mice were given PBS at both immunizations. Humoral immune responses were assessed by ELISA or pseudovirus neutralization assay over time. FIGS. 46B-46D Binding ELISA over time to wildtype RBD FIG. 46B, BA.4 / 5 RBD FIG. 46C, and JN. 1 RBD FIG. 46D. FIG. 46E Neutralization titer against JN.l pseudovirus. Graphs show geometric mean with geometric SD, n=9-10 mice per group. Black arrows represent prime and boost timepoints.

[0072] FIGS. 47A and 47B illustrate SARS-CoV-2 FLiRT spike DNA-LNP induces specific neutralizing antibody responses. FIG. 47A Layout of immunization scheme. BALB / c mice were vaccinated twice separated by 3 weeks. Mice were primed with wild-type BNT162b2 (0.2ug) and boosted with either the wild-type / BA.4 / 5 bivalent BNT162b2 vaccine (2ug) or JN.l spike DNA-LNP (2ug) or FLiRT spike DNA-LNP (2ug). Humoral immune responses were assessed by pseudovirus neutralization assay over time. FIG. 47B Neutralization titer against KP3.2 pseudovirus. Graphs show geometric mean with geometric SD, n=5 mice per group.

[0073] FIGS. 48A through 48C illustrate self-assembling nanoparticle immunogen is immunogenic using the DNA-LNP platform. BALB / c mice were immunized with 0.5ug of the GT8-60mer DNA-LNP. Germinal center populations were assessed in the draining lymph nodes 7 days post immunization. FIG. 48A Tfh cell responses. Pre-gated on live CD 19- CD4+ CD44+. FIG. 48B Germinal center B cell responses. Pre-gated on live CD4- CD19+. FIG. 48C GT8-specific GC B cell responses. Pre-gated on live CD4- CD19+ CD38- Fas+. Dots represent individual mice, n=5 mice per group. Plots show geometric mean with geometric SD.

[0074] FIGS. 49A and 49B illustrate kinetics of in vivo antigen expression. FIG. 49A Kinetics of luciferase expression over time quantified using IVIS. FIG. 49B Images of representative timepoints (4 hours, 1 day, 12 days, and 40 days). 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.

[0075] FIG. 50 illustrates hemagglutinin head-specific monoclonal antibody 2-12C DNA-LNP generated robust antibody expression in vitro. Human IgG was quantified from supernatant of cultured Expi 293T cells transfected with DNA expressing 2-12C mAb delivered using Expifectamine, formulated as a DNA-LNP, or Expifectamine-complexed empty vector control pVAX or GFP. DETAILED DESCRIPTION

[0076] The present disclosure relates to lipid nanoparticles (LNP) as well as compositions comprising an LNP or plurality of LNPS. In some embodiments, the compositions comprise at least one lipid of the present disclosure and at least one helper lipid. In some embodiments, the invention provides a composition comprising at least one lipid or LNP for delivery of DNA molecules into cells.

[0077] Thus, in various embodiments, the invention relates to methods of gene delivery using the composition comprising at least one lipid or LNP. In some embodiments, the invention provides a composition comprising at least one lipid or LNP for preventing or treating various diseases or disorders in a subject in need thereof.

[0078] Definitions

[0079] 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 invention 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.

[0080] As used herein, each of the following terms has the meaning associated with it in this section.

[0081] 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.

[0082] “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.

[0083] “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 (Cl-Cl2 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 dimethyl ethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-l-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., Cl-6 means one to six carbon atoms) and includes straight, branched chain, or cyclic substituent groups.

[0084] 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(Cl-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.

[0085] “Alkylene” or “alkylene chain” refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a radical group, consisting solely of carbon and hydrogen, which is saturated 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 (Cl-Cl5 alkylene),one to twelve carbon atoms (Cl-Cl2 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.

[0086] “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, norbomyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like.

[0087] Unless specifically stated otherwise, a cycloalkyl group is optionally substituted.

[0044] “Cycloalkylene” is a divalent cycloalkyl group. Unless otherwise stated specifically in the specification, a cycloalkylene group may be optionally substituted.

[0088] 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 heteroalkyl group 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.

[0089] “Heterocyclyl” or “heterocyclic ring” refers to a stable 3- to 18-membered nonaromatic 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,

[0090] 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[l,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl,

[0091] 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.

[0092] 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 7t (pi) electrons, where n is an integer.

[0093] 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.

[0094] 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 quatemized. 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.

[0095] 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-l,3-dioxepin and hexamethyl eneoxi de.

[0096] 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.

[0097] 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.

[0098] The aforementioned listing of heterocyclyl and heteroaryl moi eties is intended to be representative and not limiting.

[0099] 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.

[00100] 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.

[00101] 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.

[00102] 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.

[00103] 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 nonhydrogen atoms such as, but not limited to: a halogen atom such as F, Cl, Br, and I; oxo groups (=0); hydroxyl groups (-OH); alkoxy groups (-ORa, where Ra is Cl-Cl2 alkyl or cycloalkyl); carboxyl groups (-OC(=O)Ra or -C(=O)ORa, where Ra is H, Cl-Cl2 alkyl or cycloalkyl); amine groups (-NRaRb, where Ra and Rb are each independently H, Cl-Cl 2 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.

[00104] 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.

[00105] 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.

[00106] As used herein, “nucleic acid” is meant to include any nucleic acid, whether composed of deoxyribonucleosides or ribonucleosides, and whether composed of phosphodi ester 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.

[00107] “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.

[00108] 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.

[00109] 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.

[00110] The term “DNA” as used herein is defined as deoxyribonucleic acid.

[00111] The term “RNA” as used herein is defined as ribonucleic acid.

[00112] “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 sequence of 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.

[00113] “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.

[00114] “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.

[00115] 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.

[00116] “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.

[00117] 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.

[00118] 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).

[00119] 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.

[00120] The term “pharmaceutically acceptable” as used herein refers to approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[00121] 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.

[00122] In certain instances, the polynucleotide or nucleic acid of the invention 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 one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

[00123] 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.

[00124] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA or RNA methods.

[00125] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[00126] The term “recombinant RNA” as used herein is defined as RNA produced by joining pieces of RNA from different sources.

[00127] As used herein, the terms “subject,” “individual,” “host,” and “patient,” are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. The methods described herein are applicable to both human therapy and veterinary applications. In some embodiments, the subject is a mammal, and in other embodiments the subject is a human.

[00128] As used herein, “patient in need thereof’ or “subject in need thereof’ refers to a living organism suffering from or prone to a disease or condition that can be treated by administration of at least one composition, vaccine or pharmaceutical composition disclosed herein, including, for example, a vaccine comprising a nucleic acid sequence encoding a antigen, such as a nucleic acid sequence that encodes a viral antigen according to the methods described herein. A “patient in need thereof’ or “subject in need” may also refer to a living organism that is receiving a DNA vaccine (or pharmaceutical composition comprising a neoantigen DNA vaccine), or has received a DNA vaccine (or pharmaceutical composition comprising a neoantigen DNA vaccine); or has a tumor or cancer. Non-limiting examples include humans, other mammals, such as bovines, rats, mice, dogs, monkeys, goat, sheep, cows, deer, and other non-mammalian animals. In embodiments, a patient in need thereof or subject in need thereof is human. In some embodiments, the subject in need thereof is a human patient that is suspected of having cancer or has been diagnosed with cancer and exhibits.

[00129] As used herein, the term “identical” refers to two or more sequences or subsequences which are the same.

[00130] 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 in both 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 negativescoring 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, 58735787, 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 indication of 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.

[00131] 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.

[00132] “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 occurring variants 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.

[00133] 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.

[00134] 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 aldehyde and 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.

[00135] 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.

[00136] 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.

[00137] 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.

[00138] 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.

[00139] 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 crossspecies 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.

[00140] 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 a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[00141] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen-specific adaptive immune response.

[00142] 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.

[00143] 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.

[00144] 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 hyperplasias, neoplasias, 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 kidney or 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).

[00145] “Cancer,” as used herein, refers to the abnormal growth or division of cells.

[00146] 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.).

[00147] An “effective amount” as used herein, means an amount that is therapeutically effective, prophylactically effective, or both.

[00148] 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.

[00149] 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 therapeutically effective 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.

[00150] 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.

[00151] 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.

[00152] 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.

[00153] 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.

[00154] 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.

[00155] The phrase “under transcriptional control” or “operatively linked” asusedherein 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.

[00156] 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.

[00157] “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.

[00158] Ranges: throughout this disclosure, various aspects of the invention 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 invention. 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.

[00159] The present disclosure relates, in part, to lipid nanoparticles (LNP) as well as compositions thereof, and methods of use thereof to deliver DNA molecules to a nucleus of a cell. This disclosure also relates, in part, to the discovery that said LNP, and / or compositions thereof, delivered DNA molecules to the nucleus with enhanced efficiency and low toxicity. Thus, in some aspects, the present disclosure also relates to the method of delivering DNA molecules into targets (e.g., cells) using the LNP compositions. In various embodiments, the invention relates to methods of gene delivery 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 encoding an agent for the treatment of the disease or disorder or encoding an antigen for inducing an immune response against an antigen associated with the disease or disorder.

[00160] Lipid Nanoparticles (LNP)

[00161] The present disclosure relates, in part, to lipid nanoparticle compositions (LNP) comprising a combination of one or more lipid compounds. In some embodiments, the LNP comprises at least one ionizable lipid compound.

[00162] 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.

[00163] In some embodiments, the lipid nanoparticles have a mean diameter of from about 30 nanometers (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, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm.

[00164] 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, the lipid 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.

[00165] 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 pg / mL with lipid containing ethanol at a volumetric ratio of 1:3 (ethanol: citrate buffer) using microfluidic mixing device.

[00166] In various embodiments, the lipids or the LNP of the present disclosure are substantially non-toxic.

[00167] 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 lipids of 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%.

[00168] 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.

[00169] 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-(l- (2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,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-(l-(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.).

[00170] In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention 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), l,2-dilinoleyoxy-3-morpholinopropane (DLin-MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1), 1,2- dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-l,2- propanediol (DLinAP), 3-(N,N-dioleylamino)-l,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2- N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA).

[00171] 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%.

[00172] 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.

[00173] 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%.

[00174] 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 some embodiments, 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%.

[00175] 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, l-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.

[00176] 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%.

[00177] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof.

[00178] 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%.

[00179] 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

[00180] 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%.

[00181] 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.

[00182] Exemplary neutral lipids include, for example, distearoylphosphatidyl choline (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG, distearoyl-phosphatidylethanolamine (DSPE)-maleimide-PEG2000, 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, l-stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), stearoyloleoylphosphatidylcholine (SOPC), and l,2-dielaidoyl-sn-glycero-3-phophoethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

[00183] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[00184] In some embodiments, the LNP comprises one or more steroid. A “steroid” is a compound comprising the following carbon skeleton:

[00185] In some embodiments, the steroid or steroid analogue is cholesterol.

[00186] 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.

[00187] 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 -(monom ethoxy -polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[00188] 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 some embodiments, the polyethylene glycol-lipid is N-[(methoxy poly(ethylene glycol)2000)carbamyl]-l,2-dimyristyloxlpropyl-3- amine (PEG-c-DMA). In some embodiments, the polyethylene glycollipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-0-(2’,3’-di(tetradecanoyloxy)propyl-l-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S- DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co- methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

[00189] 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 some embodiments, the additional lipid is present in the LNP in an amount from about 1 mol% to about 5 mol%. In some embodiments, one or more additional lipid is present in the LNP in about 1 mol% or about 2.5 mol%.

[00190] 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.

[00191] 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).

[00192] In some embodiments, a derivative of SM-102 is a compound of Formula (II): Formula (II) wherein n is from about 1 to about 3; and wherein Ri is an alkyl or heteroalkyl group comprising from about 7 to about 25 carbon atoms. In some embodiments, Ri comprises 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 Ri comprises two non-contiguous alkyl or heteroalkyl groups covalently bound to a COO group. In some embodiments n is 1 or 2, and Ri comprises a first and a second noncontiguous 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 Ri is a C12 to C28 monoglyceride, alkenyl, alkyl, aryl, or aralkyl.

[00193] 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 II, wherein Ri is chosen from one of the following: o

[00194] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:

[00195] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:

[00196] 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 some embodiments, the targeting moiety is a ligand which directs the LNP to a receptor found on a cell surface.

[00197] In some embodiments, the composition comprises one or more internalization domains. For example, In some embodiments, the composition comprises one or more domains which bind to a cell to induce the internalization of the LNP. For example, In some embodiments, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP.

[00198] LNP Compositions

[00199] In various aspects, the present disclosure also provides compositions comprising the LNP described herein. In various embodiments, the composition comprises one or more nucleic acid molecules. In some embodiments, one or more nucleic acid molecules are encapsulated within the lipid.

[00200] In some embodiments, 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.

[00201] 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 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.

[00202] 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 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 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 disclosed molar 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.

[00203] 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.

[00204] 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.

[00205] In some embodiments, the composition is free of a molecule of Formula I: or a derivative thereof.

[00206]

[00207] 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 invention include, but are not limited to, cytokines, chemokines, costimulatory molecules, enzymes, antibodies, antigens, or any combination thereof.

[00208] In some embodiments, the DNA molecule comprises a promoter or regulatory sequence. In some embodiments, 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 some embodiments, the DNA molecule of the invention comprises an expression vector, and the invention comprises a method for the introduction of exogenous DNA into the nucleus of cells or tissues of interest with concomitant expression of the exogenous DNA in the cells or tissues of interest.

[00209] In some embodiments, the composition comprises a DNA molecule encapsulated within the LNP. In various embodiments, the compositions comprising a DNA molecule encapsulated within the LNP have particular advantages over mRNA-LNPs, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation.

[00210] 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%.

[00211] In some embodiments, the fraction is about less than about 5%. In some embodiments, the fraction is about less than about 3%. In some embodiments, the fraction is about less than about 1%. In some embodiments, the fraction is about less than about 2%. In some embodiments, the fraction is about less than about 4%. In some embodiments, the fraction is about less than about 6%. In some embodiments, the fraction is about less than about 8%. In some embodiments, the fraction is about less than about 10%. In some embodiments, the fraction is about less than about 12%. In some embodiments, the fraction is about less than about 15%. In some embodiments, the fraction is about less than about 20%. In some embodiments, the fraction is about less than about 30%. In some embodiments, the fraction is about less than about 40%. In some embodiments, the fraction is about less than about 50%. In some embodiments, the fraction is about less than about 60%. In some embodiments, the fraction is about less than about 70%.

[00212] In some embodiments, about 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 about 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%.

[00213] 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 about 10%. In some embodiments, the fraction is about less than about 5%. In some embodiments, the fraction is about less than about 3%. In some embodiments, the fraction is about less than about 1%. In some embodiments, the fraction is about less than about 2%. In some embodiments, the fraction is about less than about 4%. In some embodiments, the fraction is about less than about 6%. In some embodiments, the fraction is about less than about 12%. In some embodiments, the fraction is about less than about 15%. In some embodiments, the fraction is about less than about 20%. In some embodiments, the fraction is about less than about 30%. In some embodiments, the fraction is about less than about 40%. In some embodiments, the fraction is about less than about 50%. In some embodiments, the fraction is about less than about 60%. In some embodiments, the fraction is about less than about 70%.

[00214] In some embodiments, the DNA does not activate any pathophysiologic pathways, 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 a DNA molecule encapsulated within the LNP induces greater production of antigen-specific antibody production as compared to antigen encoded by an mRNA molecule.

[00215] In some embodiments, the nucleic acid molecule encodes a therapeutic agent. In some embodiments, the nucleic acid molecule encodes a plurality of therapeutic agents. In some embodiments, the therapeutic agent is an antigen. In some embodiments, the DNA encodes one or more antigens. In some embodiments, the therapeutic agent is a binding molecule (e.g., an antibody or antibody fragment) specific for binding to an antigen. In some embodiments, the DNA encodes one or more binding molecules.

[00216] In various 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 invention includes a nucleic acid molecule encoding an adjuvant.

[00217] In some embodiments, the antigen is encoded by a nucleotide sequence of a nucleic acid molecule. In some embodiments, 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 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.

[00218] In some embodiments, the composition comprises a nucleic acid sequence which encodes an antigen. For example, in some embodiments, the composition comprises a 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.

[00219] 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 some embodiments, 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.

[00220] 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. For example, In some embodiments, the amino acid sequence of the antigen has a degree of identity with respect to the original amino acid sequence of at least about 60%, advantageously of at least about 70%, preferably of at least about 85%, and more preferably of at least about 95%.

[00221] Viral Antigens

[00222] 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, Picomaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In some embodiments, the viral antigen is from papilloma viruses, for example, human papilloma 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.

[00223] SARS-CoV-2 Antigen

[00224] In some embodiments, 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).

[00225] In some embodiments, the SARS-CoV-2 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 SARS-CoV-2 virus.

[00226] Hepatitis Antigen

[00227] In some embodiments, 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.

[00228] In some embodiments, 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.

[00229] In some embodiments, 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., El 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.

[00230] In some embodiments, 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.

[00231] In some embodiments, 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.

[00232] In some embodiments, 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 F core 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.

[00233] Human Papilloma Virus (HPV) Antigen

[00234] In some embodiments, 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 some embodiments, 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 with out oncogenic activity, or fragments, or variant thereof from any HPV type. In some embodiments the vaccine includes El or E2 or E5 or fragments and variants thereof.

[00235] RSV Antigen

[00236] In some embodiments, 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 some embodiments, 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 some embodiments, 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 invention, In some embodiments, the RSV F protein is in a prefusion form or a postfusion form.

[00237] In some embodiments, 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 some embodiments, the antigen comprises a RSV G protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23993). In some embodiments, 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 Hl 123, or a fragment or variant thereof.

[00238] In other embodiments, the RSV antigen comprises a human RSV non-structural protein 1 (“NS1 protein”), or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV NS1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23987.1). In some embodiments, the RSV antigen comprises RSV non-structural protein 2 (“NS2 protein”), or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV NS2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23988.1). In some embodiments, the RSV antigen comprises human RSV nucleocapsid (“N”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen is RSV N protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23989.1). In some embodiments, the RSV antigen comprises human RSV Phosphoprotein (“P”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV P protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23990.1). In some embodiments, the RSV antigen comprises human RSV Matrix protein (“M”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV M protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23991.1).

[00239] In still other embodiments, the RSV antigen comprises human RSV small hydrophobic (“SH”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV SH protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23992.1). In some embodiments, the RSV antigen comprises human RSV Matrix protein2-l (“M2-1”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV M2-1 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23995.1). In some embodiments, the RSV antigen comprises RSV Matrix protein 2-2 (“M2-2”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV M2-2 protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23997.1). In some embodiments, the RSV antigen comprises RSV Polymerase L (“L”) protein, or fragment or variant thereof. For example, In some embodiments, the RSV antigen comprises RSV L protein, or fragment or variant thereof, from the RSV Long strain (GenBank AAX23996.1).

[00240] Influenza Antigen

[00241] 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 HAI, 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 Hl, influenza A serotype H3, or influenza B.

[00242] 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. The embodiment would include self assembling DNA assembling multivalent structures displaying the RBD of HA on the multivalent scaffold encoded in the DNA for invivo expression ( Konrath et al cell reports 2022). Konrath et al 2024 Vaccine submitted).

[00243] In some embodiments, the influenza antigen comprises Hl HA, H2 HA, H3 HA, H5 HA, H7 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.

[00244] Human Immunodeficiency Virus (HIV) Antigen

[00245] In some embodiments, the antigen comprises an HIV antigen or fragment thereof, or variant thereof.

[00246] 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 gpl20, gp41, or a combination thereof.

[00247] 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.

[00248] 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 I, or subtype K.

[00249] Parasite Antigens

[00250] 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 (eg., pinworms). In some embodiments, the ectoparasite is lice, fleas, ticks, and mites.

[00251] 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.

[00252] 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.

[00253] Malaria Antigen

[00254] 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 some embodiments, the antigen comprises an antigen from a parasite causing malaria. In some embodiments, the malaria causing parasite is Plasmodium falciparum.

[00255] In some embodiments, the malaria antigen comprises one or more of P. falciparum immunogens CS; LSA1; TRAP; CelTOS; and Amal. The immunogens may be full length or immunogenic fragments of full-length proteins.

[00256] Bacterial Antigens

[00257] 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.

[00258] 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.

[00259] 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.

[00260] Mycobacterium tuberculosis Antigens

[00261] 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.

[00262] Fungal Antigens

[00263] 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.

[00264] Tumor Antigens

[00265] 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, nonHodgkin'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.

[00266] Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. In some embodiments, 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 invention.

[00267] Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), P-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RUI, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M- CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, 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.

[00268] 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, CD 19, CD20, idiotype) have been used as targets for passive immunotherapy with monoclonal antibodies with limited success.

[00269] The type of tumor antigen referred to in the invention may also be a tumorspecific 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.

[00270] Non-limiting examples of TSA or TAA antigens include the following: Differentiation antigens such as MART-l / MelanA (MART-I), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2 and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; 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, pl85erbB2, pl80erbB-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, M0V18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

[00271] Exemplary antigens that can be delivered using the LNPs of the invention 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.

[00272] 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.

[00273] In some embodiments, the 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.

[00274] In some embodiments, the DNA molecule encodes a biologically active molecule. Exemplary biologically active molecules that can be encoded include, but are not limited to, cytokines, chemokines, growth factors, costimulatory molecules, monoclonal antibodies, protein hormones and bispecifics and derivatives and enzymes.

[00275] 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 invention. Therefore, the scope of the present disclosure includes nucleotide sequences that are substantially homologous to a wild-type or parental nucleotide sequence.

[00276] 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.

[00277] In some embodiments, the invention 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 invention relates to a construct, comprising a nucleotide sequence encoding an adjuvant.

[00278] Exemplary adjuvants include, but is not limited to, alpha-interferon, gammainterferon, platelet derived growth factor (PDGF), TNFa, TNF0, 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, 0x40, 0x40 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. The amino acid sequence of CD40: MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNL HEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREAS SQ APFIASLCLKSPGRFERILLRAANTHS S AKPCGQQ SIHLGGVFEL QPGASVFVNVTDPSQVSHGTGFTSFGLLKL The amino acid sequence of CD80: MGHTRRQGTSPSKCPYLNFFQLLVLAGLSHFCSGVIHVTKEVKEVATLSCGHNVSVE ELAQTRIYWQKEKKMVLTMMSGDMNIWPEYKNRTIFDITNNLSIVILALRPSDEGTY EC VVLKYEKD AFKREHL AEVTL S VKADFPTPSISDFEIPT SNIRRIIC ST SGGFPEPHLS WLENGEELNAINTTVSQDPETELYAVSSKLDFNMTTNHSFMCLIKYGHLRVNQTFN WNTTKQEHFPDNLLPSWAITLISVNGIFVICCLTYCFAPRCRERRRNERLRRESVRPV The amino acid sequence of CD86: MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFW QDQENL VLNE VYLGKEKFD S VHSK YMGRT SFD SD S WTLRLHNLQIKDKGL YQCIIH HKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLL RTKNSTIEYDGVMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPF SIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMERE ESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF

[00279] 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.

[00280] 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 some embodiments, 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.

[00281] In some embodiments, the construct is operatively bound to one or more regulatory sequence for the expression of the nucleotide sequence of the invention, 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.

[00282] In some embodiments, the composition of the invention comprises a DNA vector or plasmid for expression of an encoded nucleotide sequence. In some embodiments, the composition of the invention comprises a 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 some embodiments, the composition comprises an DNA expression plasmid encapsulated within a LNP. In certain instances, the LNP enhances cellular and nuclear uptake of the DNA expression plasmid.

[00283] In some embodiments, the nucleic acid molecule is a DNA plasmid comprising a nucleotide sequence encoding a therapeutic agent, an antigen, an antibody, a fragment of antibody or a gene. Thus, In some embodiments, the composition of the present disclosure comprises a DNA plasmid comprising a nucleotide sequence encoding an antigen. In some embodiments, the composition of the invention comprises a DNA plasmid comprising a nucleotide sequence encoding an antibody, or an antibody fragment. In some embodiments, the composition of the invention comprises a DNA plasmid comprising a nucleotide sequence encoding an adjuvant. In some embodiments, the composition of the invention comprises a DNA

[00284] plasmid comprising a nucleotide sequence encoding one or more antigens and one or more adjuvants.

[00285] The DNA can be produced using methods known in the art. 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.

[00286] In some embodiments, a desired expression construct is generated using PCR. In some embodiments, 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 some embodiments, the DNA is a full length gene of interest of a portion of a gene. The gene can include some or all of the 5' and / or 3' untranslated regions (UTRs). The gene can include exons and introns. In some embodiments, 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.

[00287] The nucleic acid sequences coding for the agent of the invention 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.

[00288] Alternatively, the gene of interest can be produced synthetically.

[00289] 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.

[00290] 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).

[00291] 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 the nucleic acids into a host cell (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 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.

[00292] In some embodiments, the nucleic acid molecule, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

[00293] 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.

[00294] In various 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.

[00295] In some embodiments, the transfection reagent forms a liposome. Liposomes, In some embodiments, increase intracellular stability, increase uptake efficiency and improve biological 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.

[00296] In some embodiments, the delivery vehicle comprising the DNA molecules are administered directly to the subject, in vivo. In some embodiments, in vivo delivery of DNA molecules using the LNP of the invention stimulates CD8 immune responses.

[00297] LNP Vaccine

[00298] In some embodiments of the invention, 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 (eg., 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.

[00299] In the context of the present disclosure, the term “vaccine” refers to a substance that induces immunity upon inoculation into animals.

[00300] 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 antigen might also be formulated with an adjuvant. 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. In another non-limiting example, a vaccine may comprise one or more adjuvants. 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.

[00301] 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.

[00302] 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.

[00303] 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 invention, 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 contact with 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.

[00304] 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.

[00305] 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.

[00306] 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.

[00307] 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 Thl, Th2, Th9, Thl7, 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 invention, the Tfh subtype provides help in the generation of high affinity antibodies. Targeting Domain

[00308] In some embodiments, the invention comprises targeted delivery of a DNA molecule to a specific cell type of interest. In some embodiments, therefore the LNP composition of the invention comprises a targeting domain bound or conjugated to one or more lipid of the LNP. In some embodiments, the targeting domain of the instant invention 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.

[00309] 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.

[00310] In some embodiments, the targeting domain of the invention 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.

[00311] 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.

[00312] 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 on several 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 fulLlength 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 pharmacokinetic activity may be generated in a bacterial expression system. Single chain Fv fragments show low immunogenicity. Peptide targeting moieties

[00313] In some embodiments, the targeting domain of the invention 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

[00314] 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.

[00315] 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.

[00316] 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 nonconserved 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 is employed 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.

[00317] Nucleic acid targeting moieties

[00318] In some embodiments, the targeting domain of the invention comprises an isolated nucleic acid (e.g., DNA or RNA), including for example, an aptamer, a DNA oligonucleotide or a RNA oligonucleotide. In some embodiments, the nucleic acid targeting domain specifically binds to a target of interest. For example, In some embodiments, the nucleic acid comprises a nucleotide sequence that specifically binds to a target of interest.

[00319] 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.

[00320] Pharmaceutical LNP Compositions

[00321] 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.

[00322] 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 the invention 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.

[00323] Pharmaceutical compositions that are useful in the methods of the invention 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.

[00324] A pharmaceutical composition of the invention 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.

[00325] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention 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.

[00326] In some embodiments, the composition comprises a DNA-LNP with a lipid to DNA weight ratio of from 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: about 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. In some embodiments, the composition comprises one or a plurality of LNPs with an average weight ratio of lipid to DNA of from about 35:1 to about 45:1. In some embodiments, the composition comprises one or a plurality of LNPs with an average weight ratio of lipid and cholesterol to DNA of from about 35:1 to about 45:1. In some embodiments, the composition comprises one or a plurality of LNPs with an average weight ratio of lipid to DNA of from about 10:1 to about 40:1. In some embodiments, the average weight ratio of lipid to DNA of all LNPs in a composition is about 40: about 1.

[00327] In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

[00328] Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

[00329] 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.

[00330] 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 some embodiments 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.

[00331] 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 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.

[00332] A pharmaceutical composition of the invention 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.

[00333] 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) of the 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).

[00334] 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 some embodiments 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.

[00335] 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.

[00336] 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 invention 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.

[00337] The therapeutic compounds or compositions of the invention 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.

[00338] Methods of Delivery

[00339] In some embodiments, the present disclosure provides a method for delivery of a nucleic acid molecule, therapeutic agent, or any combination thereof to a target of interest. According to 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.

[00340] 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.

[00341] 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.

[00342] In some embodiments, the method is a gene delivery method. Therefore, in some embodiments, the invention encompasses in vivo delivery of DNA molecules comprising genes or gene fragments.

[00343] In some embodiments, the method is a gene editing method. Therefore, in some embodiments, the invention encompasses in vivo delivery of DNA molecules encoding gene editing molecules (e.g., Cas9, sgRNA or a combination thereof).

[00344] In some embodiments, 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.

[00345] In some embodiments, the method comprises a single administration of the composition. In some embodiments, the method comprises multiple administrations of the composition.

[00346] 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.

[00347] 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.

[00348] 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 longterm 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.

[00349] 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 invention.

[00350] In some embodiments, 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.

[00351] Treatment Methods

[00352] The present disclosure provides methods of inducing an adaptive immune response in a subject in need thereof 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.

[00353] In some embodiments, the method provides immunity in the subject to an infection, cancer, or disease or disorder associated with an antigen. The present disclosure thus provides 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.

[00354] 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.

[00355] 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, nonsmall 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.

[00356] 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 some embodiments, 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 some embodiments, the composition is administered to a subject who has increased likelihood, though genetic factors, environmental factors, or the like, of developing cancer.

[00357] 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.

[00358] In some embodiments, the 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 5fold factor. In some embodiments, the adaptive immune response is increased by a 7-fold factor. 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.

[00359] 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.

[00360] In some embodiments, the 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 2fold 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 immunogenicity is 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.

[00361] 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.

[00362] In some embodiments, 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.).

[00363] 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 some embodiments, 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 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.

[00364] In some embodiments, the method comprises administering to subj ect a plurality of nucleic acid molecules encoding a plurality of antigens, adjuvants, or a combination thereof.

[00365] In some embodiments, the method of the invention 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.

[00366] 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.

[00367] Administration of the compositions of the invention in a method of treatment can be achieved in a number of different ways, using methods known in the art. In some embodiments, the method of the invention 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 some embodiments, the method comprises subcutaneous delivery of the composition. In some embodiments, the method comprises inhalation of the composition. In some embodiments, 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.

[00368] It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

[00369] The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions encoding an antigen, adjuvant, or a combination thereof, described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from ng / kg / day and 100 mg / kg / day. In some embodiments, the invention envisions administration of a dose which results in a concentration of the compound of the present disclosure from lOnM and 10 DM in a mammal.

[00370] Typically, dosages which may be administered in a method of the invention to a mammal, preferably a human, range in amount from 0.01 pg 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 pg to about 10 mg per kilogram of body weight of the mammal. More preferably, the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.

[00371] 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.

[00372] In some embodiments, administration of the composition or vaccine of the present disclosure may be performed by single administration or boosted by multiple administrations.

[00373] In some embodiments, the invention 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.

[00374] In some embodiments, 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.

[00375] The disclosure also relates to a method of inducing proliferation of Natural Killer (NK) cells in a subject in need thereof comprising administering to the subject one or more formulations disclosed herein. In some embodiments, the disclosure also relates to a method of stimulating RUNX3, CDK2ap2 and TRAF1 in NK cells comprising administering to the subject one or more formulations disclosed herein. EXAMPLES

[00376] 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 invention 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.

[00377] 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 utilize the 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

[00378] 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 protein in mice. As proof-of-concept, a lipid nanoparticle formulation of 50:38.5:10:1.5 (SM- 102:Cholesterol:DSPC:DMG-PEG) was utilized to encapsulate plasmid DNA for in vivo delivery similar to those used in licensed SARS-CoV-2 mRNA-LNP vaccines. Lipid nanoparticle formulation of plasmid DNA was capable of inducing robust antigen-specific immunity with a unique profile compared with mRNA- LNP and adjuvanted protein. These findings are relevant for DNA vaccine platform development providing an additional delivery method with unique immune phenotypes, further expanding product applications. Nucleoside-modified mRNA-LNP vaccines have demonstrated potent immunogenicity in animals and humans, including those currently approved for SARS-CoV-2 (5, 6). The LNP component has been shown to be highly pro-inflammatory and can adjuvant nucleoside-modified mRNA as well as protein vaccines (11). This is due in part to the transient upregulation of numerous pro-inflammatory cytokines, such as IL-6, which can potentiate adaptive immune engagement (12, 46). Immunogenicity has been linked to the ionizable component, as LNPs formulated without the ionizable lipid could not successfully adjuvant a recombinant protein influenza vaccine in mice (11). LNP-formulated plasmid DNA, however, has previously not shown similar levels of in vivo immunogenicity, despite robust transfection efficiency when using a firefly luciferase-encoding construct (35). A recent study has described an ionizable LNP formulation of SARS-CoV-2 spike- expressing plasmid DNA using SM-102 was effective at inducing proinflammatory cytokines and serum responses, but did not describe cellular correlates of immunity such as GC or T cell responses (47). This study demonstrates that a single dose of ionizable lipid-containing LNP-formulated plasmid DNA encoding either SARS-CoV-2 spike or influenza virus HA protein can indeed elicit robust cellular and humoral immunity. The altered amine to phosphate ratio was controlled when formulating antigen- encoding DNA plasmids in LNPs, which demonstrated an impact on immunogenicity. An increased N / P ratio has been associated with increased in vitro gene expression for RNA (48). The weight ratio formulation of 40:1 (an N / P ratio of 10.5:1) improved antigen-specific immunity within the germinal center, implicating the lipid to DNA ratio as a modulator of in vivo immunogenicity with different nucleic acid-LNP formulations. Based on these data, continued study of varied N / P ratios for LNP-formulated nucleic acid vaccines is important to further tailor immunogenicity for specific applications. Antigen-related differences were observed in immune response patterns. CA09 HA plasmid DNA-LNP elicited attenuated GC effector cell frequencies compared with mRNA-LNP, although absolute cell numbers were more comparable, demonstrating increased lymphoproliferation. However, these differences in GC responses did not result in attenuated HA-specific serum antibody titer, breadth, or functionality. This observation represents an interesting decoupling of the GC with functional antibodies, as these have been demonstrated to be correlated with nucleoside-modified mRNA-LNPs (37). This could be due to plasmid DNA having longer protein production kinetics as compared with mRNA (49, 50), allowing for longer antigen exposure. Interestingly, delivery of SARS-CoV-2 spike as plasmid DNA- or mRNA-LNPs elicited similar levels of GC effector cell populations as well as cytokinesecreting CD8+ and CD4+ T cells. Serum antibody responses were competitive with spike DNA-LNP immunization at levels to spike mRNA-LNP and BNT162b2. Additionally, in the T cell compartment, HA DNA-LNPs elicited superior CD8+ T cell responses compared to mRNA-LNP and adjuvanted protein vaccines. These data were largely similar when comparing T cell responses using full-length SARS-CoV-2 spike, which elicited overall comparable CD8+ T cell responses in both platforms but a bias toward DNA-LNPs in IFNy- secreting CD4+ T cells. This outcome provides additional evidence for antigen-specific differences in immunogenicity between these two LNP-formulated platforms. Additional factors influencing the immunogenicity of plasmid DNA-LNP vaccines remains to be determined. Differences in plasmid DNA quality or formulation such as the degree of supercoiling and specific codon optimization strategies could influence resulting immunogenicity. As such, studies investigating the impact of each of these factors on immune outcomes appear warranted. The development of improved formulations for DNA delivery with preservation of the stability aspects of the DNA platform as we describe here represent important tools for the generation of immunity against infectious agents and pathogenic cells.

[00379] The Materials and Methods are now described

[00380] Transfection

[00381] C2C12 cells were cultured at 37°C, 5% CO2 in DMEM + 10% fetal bovine serum + 1% Penicillin / Streptomycin (D10) and seeded into 6-well plates. The following day, cells were transfected with plasmid DNA using Lipofectamine 3000 kit (Thermo Fisher) according to manufacturer’s protocol. Briefly, DNA was mixed with P3000 reagent and complexed with Lipofectamine 3000 in Opti-MEM medium for 15 minutes at room temperature. Subsequently, DNA-Lipid complexes were added to cells and incubated for 2 days before imaging was performed using an EVOS FL Digital Inverted Microscope on light and GFP fluorescence settings (470 nm excitation, 525 nm emission).

[00382] Preparation of DNA-LNPs and mRNA-LNPs

[00383] Codon optimized DNA plasmids were obtained from Genscript with the antigen in a pVAXl vector under the control of an IgE leader sequence to facilitate secretion. The construct encoding the full-length SARS-CoV-2 Spike glycoprotein (with D614G mutation) has been described previously (30-32). Full length CA09 HA was synthesized as above. mRNA vaccines encoding the full-length SARS-CoV-2 Spike glycoprotein and CA09 HA were described previously (38, 39). To create plasmid DNA-LNPs and mRNA-LNPs were formulated using microfluidic mixing of organic phase containing lipids and aqueous phase containing plasmid DNA or mRNA. In brief, lipids were dissolved in ethanol at a molar ratio of 50:38.5:10:1.5 (SM102:Cholesterol:DSPC:DMG-PEG). Lipids utilized include SM102 (Echelon Bioscience, Cat#N-l 102), Cholesterol (Avanti polar lipids, Cat#700000), DMG-PEG 2000 (Avanti polar lipids, Cat#880151), and 18:0 PC (DSPC; Avanti polar lipids, Cat#850365).The plasmid DNA or mRNA were diluted in citrate buffer (citrate buffer 50 mM, pH-4) at a concentration of 129 pg / mL and mixed with lipid containing ethanol at a volumetric ratio of 1:3 (ethanol: citrate buffer) using microfluidic mixing device (NanoAssembler Ignite, Precision Nanosystems). The LNPs were then dialysed against lx PBS buffer in 12-14 kDa dialysis membrane (Fisher Scientific Cat#08700158) for 2 h. The LNP formulations were characterized for their size, poly dispersity index and zeta potential using Zetasizer Pro ZS (Malvern Panalytical). The encapsulation efficiency of DNA and mRNA in LNPs were analyzed using Quant-iT RiboGreen assay and Quant-iT PicoGreen assay (ThermoFisher Scientific). [003 84]      Animal studies

[00385] studies were conducted under protocol 201399 approved by the Wistar Institute Institutional Animal Care and Use Committee (IACUC). Six- to eight-week-old female BALB / cJ mice (lackson Laboratory) were housed in the Wistar Institute Animal Facility. Mice were immunized with indicated DNA-LNPs or mRNA-LNPs at varying doses (2pg, 0.2pg, or 0.02pg) in the tibialis anterior muscle in a total volume of 50pL diluted in PBS. For protein vaccination, Ipg of recombinant full-length CA09 HA (Sino Biologicals Cat# 11055-VNAB) was formulated 1:1 v / v with Addavax Adjuvant (Invivogen vax-adx-10). Mice were bled by submandibular bleed biweekly for antibody titers.

[00386] Tissue processing

[00387] Mice were euthanized using CO2 at indicated timepoints. Popliteal and iliac draining lymph nodes were harvested into ice cold RPMI + 10% FBS + 1% Penicillin / Streptomycin (R10). Lymph nodes were mechanically dissocated over a 40pm strainer, followed by washing with R10. Lymph nodes were counted using a ViCell Blu Cell Viability Analyzer (Beckman Coulter) and subjected to downstream staining for flow cytometry. Spleens were harvested similarly and mechanically dissociated using a Stomacher 80 (Seward). Splenocytes were filtered using a 40pm strainer before ACK lysis for 5 minutes at room temperature. Cells were quenched by dilution with PBS and resuspended in fresh RIO before a second 40pm filtration. Splenocytes were then counted using a ViCell Blu and subjected to downstream assays.

[00388] Peptide stimulation of splenocytes

[00389] Splenocytes were plated and incubated in the presence of Protein Transport Inhibitor Cocktail (eBioscience) and overlapping peptides spanning the SARS-CoV-2 Spike protein or CA09 influenza HA protein. Peptides were 15 amino acids in length with a 9 amino acid overlap. Negative control samples were stimulated in the presence of DMSO and positive control samples in the presence of Cell Stimulation Cocktail (eBioscience) in lieu of peptides. All samples were incubated in the presence of FITC anti-mouse CD 107a (Clone 1D4B, Biolegend). Splenocytes were stimulated for 6 hours at 37°C.

[00390] Flow cytometry

[00391] Germinal centers: Lymph node suspensions were resuspended in Fixable Viability Dye eF780 (eBioscience) in 0.2% BSA in PBS (FACS) for 10 min at RT. Cells were next washed before resuspension with Biotin anti-mouse CXCR5 (Clone SPRCL5, eBioscience) for 30min at RT in FACS. Cells were subsequently washed and resuspended with a surface stain cocktail for 30min at RT containing a subset of the following antibodies or probes: BUV395 anti-mouse CD19 (Clone 1D3, BD), BUV496 anti-mouse CD4 (Clone RM4-5, BD), BV421 anti- mouse CD4 (Clone GK1.5, Biolegend), BUV805 anti-mouse CD38 (Clone 90, BD), BV421 Streptavidin (Biolegend), BV510 anti-mouse Fas (Clone Jo2, BD), BV605 anti-mouse CD44 (Clone IM7, Biolegend), PE-Cy7 anti-mouse PD-1 (Clone 29F.1A12, Biolegend), FITC- conjugated CA09 HA (in house), APC-conjugated CA09 HA (in house), PE-conjugated SARS- CoV-2 Spike (in house), or FITC-conjugated SARS-CoV-2 Spike (in house). Cells were washed and resuspended in FACS buffer before acquiring on a FACSymphony A3 or A5 SE analyzer (BD). FCS files were exported and analyzed using FlowJo (Treestar).

[00392] T cell intracellular cytokine staining: After peptide stimulation, cells were first washed in PBS before incubation with Zombie Aqua (Biolegend) for lOmin at RT. Cells were washed in FACS and resuspended in a surface stain cocktail for 30min at RT containing a subset of the following antibodies: BUV496 anti-mouse CD4 (Clone RM4-5, BD), BUV805 anti-mouse CD62L (Clone MEL-14, BD), BV421 anti-mouse CD4 (Clone GK1.5, Biolegend), BV605 anti- mouse CD44 (Clone IM7, Biolegend), and APC-Cy7 anti-mouse CD8a (Clone 53-6.7, Biolegend). Cells were washed in FACS and then fixed / permeabilized using BD CytoFast / CytoFix (BD) according to manufacturer’s protocol for 20min at 4°C. Cells were washed in lx Perm / Wash before stained with a cocktail of intracellular antibodies for 30min at 4°C: BUV395 anti-mouse CD3e (Clone 145-2C11, BD), PE anti-mouse TNFot (Clone MP6-XT22, Biolegend), PE-Cy7 anti-mouse IL-2 (Clone JES6-5H4, Biolegend), and APC antimouse IFNy (Clone XMG1.2, Biolegend). Cells were resuspended in FluoroFix buffer (Biolegend) and stored at 4°C until acquisition as above.

[00393] Antigen-specific B cell probes

[00394] B cell probes were generated via direct conjugation of fluorochromes to recombinant SARS-CoV-2 Spike (Cat# 40589-V08B1, Sino Biologicals) or influenza CA09 HA (Cat# 11055-V08B1, Sino Biologicals). Lightning Link Kits for FITC (abl02884), APC

[00395] (ab201807), or PE (ab 102918) were used according to manufacturer’s protocol. Probes were stored at 4°C until use.

[00396] ELISA

[00397] 96-well half area plates (Corning, Ref# 3690) were coated in PBS overnight at 4°C with Ipg / mL of one of the following recombinant proteins where specified: SARS-CoV-2 wild type Spike RBD (Cat# 40592-V08H, Sino Biologicals), SARS-CoV-2 D614G Spike (Cat# 40589-V08H8, Sino Biologicals), SARS-CoV-2 B.l.617.2 Spike (Cat# 40589-V08H10, Sino Biologicals), SARS-CoV-2 B. 1.1.529 Spike (Cat# 40589-V08H26), SARS-CoV-2 BA.2 Spike (Cat# 40589-V08H28, Sino Biologicals), H1N1 A / Califomia / 04 / 2009 HA protein (Cat# 11055-V08H, Sino Biologicals), A / Michigan / 45 / 2015 HA (Cat# IT-003-00105ATMp, Immune Technology), A / Wisconsin / 588 / 2019 HA (Cat# IT-003-00117ATMp, Immune Technology), A / Sydney / 5 / 2021 HA (Cat# IT-003-00119ATMp, Immune Technology), or A / Victoria / 4897 / 2022 HA (Cat# IT-003-00120ATMp). The following day, plates were washed 4 times with 0.05% PBS-T before being blocked with lx DPBS containing 5% non-fat dry milk (LabScientific) and 0.2% Tween-20 (Fisher) for 1 hour at RT. After washing, plates were subsequently incubated with mouse sera serially diluted in 1% newborn calf serum and 0.2% Tween-20 in lx DPBS for 2 hours at RT before washing and incubation with 1:10000 HRP-conjugated Goat anti-mouse IgG H+L (Cat# A90-216P, Bethyl) for 1 hour at RT. Following this, plates were washed a final time and developed with 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo, Ref: 34029) for 5 minutes at RT before being stopped with 2N H2SO4. Plates were read with the BioTEK Synergy 2 plate reader and absorbance values measured at 450nm were subtracted with 570nm background OD values. Endpoint titers were calculated against naive mouse serum. The endpoint titer was defined as the highest dilution where the OD value was greater than cutoff determined using the following formula: Average (Naive Mice) + (4* SD (Naive Mice)).

[00398] Pseudovirus neutralization assay

[00399] Pseudotyped SARS-CoV-2 was produced in HEK293T cells via transient transfection using GeneJammer (Agilent) at a 1:1 ratio of IgE-SARS-CoV-2 Spike plasmid (Genscript) and pNL4-3.Luc.R-E plasmid (NID AIDS reagent). After 48 hours, transfection supernatant was harvested and enriched with FBS to 12% final volume and stored at -80°C until use. For the SARS-CoV-2 pseudovirus neutralization assay, huCHOAce2 cells (Cat# VCeL-WybO19; Creative Biolabs) were seeded into 96-well plates in DMEM + 10% FBS + 1% Penicillin / Streptomycin (D10) overnight at 37°C. The next day, mouse serum was heat inactivated, serially diluted, and incubated with SARS-CoV-2 pseudovirus for 90 min at RT, and subsequently incubated with plated huCHOAce2 cells for 72 hours. Cells were then lysed using the Britelite plus Luminescence reporter gene assay system (Cat# 6066769; Perkin Elmer) and relative luciferase units (RLU) were read using a BioTek synergy plate reader. Pseudovirus neutralization titers (ID50) were reported as the reciprocal serum titer at which RLUs were 50% reduced compared to RLUs in virus control well after background subtraction.

[00400] Hemagglutinin Inhibition Assay (HAI)

[00401] Sera were first treated with receptor destroying enzyme (RDE, Denko Co.) at 1:3 ratio overnight at 37°C. RDE-treated sera were then heat inactivated at 56°C for 45 min, followed by preadsorption with 10% Turkey red blood cells (RBCs, Lampire Biologicals) for 1 hour at 4°C with gentle agitation. Samples were applied to V-bottom 96-well plates at starting dilution neat and 2-fold serial dilution in 0.85% saline. Samples were then incubated with 4 hemagglutinating units of virus (A / California / 07 / 2009) and 0.5% Turkey RBCs in saline at room temperature for 30 min. HAI titers were scored using drip method. [00402 /      Statistics

[00403] All statistical tests were performed using GraphPad Prism 10. Graphs and error bars represent geometric means ± geometric SD. Unpaired one-way ANOVA with Bonferroni corrections were used to compare groups. The number of samples in each graph is notated in the FIG. legend. In all datasets, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[00404] The experimental results are described [00405J     Relationship between lipid components andDNA impacts immunogenicity

[00406] Plasmid DNA vaccines are typically delivered intradermally or intramuscularly. As proof-of-concept to demonstrate that lipid formulation could efficiently deliver plasmid to muscle cells, we transfected C2C12 mouse myoblasts with plasmid DNA encoding green fluorescent protein (GFP). It was observed that GFP plasmid DNA could be efficiently delivered to mouse myoblasts via lipid-based transfection (FIGS. 7A-7B).

[00407] LNP-formulated DNA is likely impacted by the association between lipid amine groups to nucleic acid backbone phosphates (N / P ratio). Plasmid is double stranded (having many more phosphate groups) and significantly longer sequences compared to mRNA expression cassettes, which could impact its relationship with the lipid components when formulated. This study sought to account for the altered amine to phosphate ratio when formulating antigen-encoding DNA plasmids and utilized multiple N / P ratios for plasmid DNA- LNP formulation. First, it was explored whether different lipid to DNA ratios impact biophysical parameters. A full-length CA09 HA expressing plasmid was formulated within LNPs at a 40:1, 20:1, and 10:1 lipid to DNA weight ratios containing the SM-102 ionizable lipid used in mRNA- 1273. Encapsulation efficiency was similar between 40:1 and 20:1 but lower for 10:1, approaching 80%. Additionally, 10:1 ratio formulation particles were larger in size with a lower (more anionic) zeta potential. Nanoparticles with a zeta potential between -10 and 10 mV are considered neutral and associated with less cell wall destruction-associated toxicity seen with cationic particles (36). To see whether this relationship could impact immunogenicity via intramuscular delivery, mice were immunized with 2pg of plasmid DNA expressing full-length CA09 HA formulated within ionizable LNPs at different lipid to DNA weight ratio formulations of 40:1, 20:1, and 10:1. As a control, empty vector DNA was additionally formulated into LNPs (pVAX DNA-LNP). The germinal center was used as an immunogenic readout, which was measured 14 days post immunization in the draining lymph nodes (DLNs). A similar Tfh response was observed with formulations at all three lipid to DNA weight ratios (FIG. 1 A). Interestingly, there was a significant attenuation in the frequency of total GC B (FIG. IB) and HA-specific GC B (FIG. IC) with a formulation at 10:1, but not 20:1. These data suggest that the relationship between lipid and DNA could impact adaptive immunity with antigen-specific B cell responses more sensitive than Tfh priming; higher lipid to DNA ratios lead to improved immune responses.

[00408] Influenza virus HA-expressing DNA-LNP elicits GC responses and functional serum antibodies

[00409] From this preliminary data, it was sought to characterize the immune response in depth after immunization with plasmid HA DNA-LNP with lipids at higher lipid to DNA ratios (40:1 weight ratio, henceforth abbreviated as DNA-LNP) in comparison with benchmark HA- encoding mRNA-LNP. First, it was sought to examine the germinal center (GC) response as LNPs are known to generate robust GCs when delivered with nucleoside-modified mRNA as well as recombinant protein (11, 37). First, antigen-specific GC responses were assessed after immunization with either HA DNA-LNP or mRNA-LNP. Mice were immunized with 2 pg of either pVAX DNA-LNP (negative control), HA DNA-LNP, or HA mRNA-LNP and GCs were assessed in the DLNs 14 days post immunization. HA DNA-LNP elicited an attenuated frequency but similar absolute numbers of GC Tfh cells (FIG. 2A) and total GC B cells compared with HA mRNA-LNP (FIG. 2B). Immunization with HA DNA-LNP generated HA-specific GC B cell responses but were attenuated in frequency and absolute number relative to HA mRNA- LNPs (FIG. 2C). Thus, these data demonstrate that DNA-LNP expressing influenza HA can elicit antigen- specific GCs.

[00410] GC reactions after mRNA-LNP immunization can be predictive of serum antibody functionality (37). Thus, we next assessed whether HA DNA-LNP could elicit serum antibody responses. Mice were immunized with 2 pg of either pVAX DNA-LNP, HA DNA-LNP or HA mRNA-LNP and serum antibody titers were assessed biweekly. As an additional control for generating influenza virus HA-specific antibody titers, we also immunized mice with recombinant CA09 HA protein formulated in Addavax, a squalene-based adjuvant similar to FDA-approved MF59. Fluad is a licensed MF59-formulated trival ent / quadrival ent flu vaccine and serves as a benchmark for influenza virus-specific antibody responses. Immunization with HA DNA-LNPs elicited comparable antibody responses over time to HA mRNA-LNP and Addavax-adjuvanted HA protein (FIG. 3A). At the final timepoint 8 weeks post immunization, HA DNA-LNP-immunized mice had significantly enhanced antibody titers relative to HA mRNA-LNP and HA protein in Addavax immunized mice (FIG. 3B). Hemagglutinin inhibition titers (HAI) against the autologous CA09 strain were robust and comparable between all three groups, demonstrating functionality of serum antibody responses (FIG. 3C). Thus, these data demonstrate that immunization with HA DNA-LNP elicits potent antibody titers comparable in titer and functionality to mRNA-LNP and protein in adjuvant formulations.

[00411] We next determined the breadth of binding antibody responses to related pandemic-lineage H1N1 influenza viruses at 8 weeks post immunization. Comparable HA-specific binding antibody titers were obtained to H1N1 A / Michigan / 45 / 2015 (FIG. 3D), A / Wisconsin / 588 / 2019 (FIG. 3E), A / Sydney / 5 / 2021 (FIG. 3F), and A / Victoria / 4897 / 2022 (FIG. 3G) with all 3 vaccine platforms. These data demonstrate that, despite an attenuated HA-specific GC B cell response with DNA-LNP (FIG. 2C), the breadth of antibody binding is similar and broad between all active vaccination groups. To address whether DNA-LNPs could elicit long lasting antibody titers, we immunized mice with 2pg of HA DNA-LNP and assessed serum antibody over through 28 weeks (7 months) post immunization. Robust antibody titers were elicited throughout the course of immunization, reaching a stable titer 8 weeks post immunization, and lasting through at least 28 weeks (FIG. 8A-8B). Thus, these data suggest that DNA-LNPs are capable of eliciting durable antibody titers for at least 7 months. Detailed studies have reported the stability of mRNA-LNP vaccine platforms over time in the context of storage conditions. Plasmid DNA has reported significant thermostability characteristics, with multiple reports describing significant cold chain-independence (1, 17, 38). However, these studies were not in reference to these LNP-formulated plasmid DNA. To address this, we immunized mice with 2pg of HA DNA-LNP stored at various temperature conditions over time. We compared DNA-LNPs that were freshly prepared, stored at 4°C for 3 months, or stored at room temperature (25°C) for 1 month for serological potency. At 4 weeks post immunization, DNA-LNPs stored at various temperature conditions all elicited comparable serum antibody titers (FIG. 3H), illustrating thermostability at various storage conditions.

[00412] Low dose HA DNA-LNP elicits robust, durable T cell responses

[00413] Next, it was explored whether HA DNA-LNP could elicit T cell responses. CD4+ T cell responses are a facet of numerous vaccination platforms and are particularly strong after immunization with mRNA and adjuvanted protein vaccines, with adjuvant choice impacting response polarization (37, 39). In the context of cytolytic T cells, nucleic acid vaccines, both DNA as well as mRNA, can generate CD8+ T cell responses superior to that of protein in adjuvant (40). Mice were immunized with 2pg of pVAX DNA-LNP, HA DNA-LNP, HA mRNA-LNP, or I pg Addavax-adjuvanted HA protein and splenic T cell responses were assessed 14 days post immunization. HA DNA-LNP demonstrated a 3-fold and 7- fold increase in IFNy- secreting CD8+ T cell responses compared with mRNA-LNP and adjuvanted protein formulations respectively (FIG. 4A). We additionally observed significantly increased CD107a- expressing (FIG. 4B) and TNFa-secreting (FIG. 4C) CD8+ T cell responses with HA DNA-LNP immunization compared both other platforms. Differing from the CD8+ T cell response, HA DNA-LNP and mRNA-LNP elicited comparable IFNy-secreting (FIG. 4D) and TNFa-secreting (FIG. 4E) CD4+ T cell responses that were significantly increased over adjuvanted protein.

[00414] However, all three immunization groups elicited similar IL-2-secreting CD4+ T cells (FIG. 4F). These data demonstrate that HA DNA-LNP formulations appear to elicit particularly robust CD8+ T cell responses with this antigen, suggesting platforms may have antigen-specific differences in immunogenicity.

[00415] Given the robust CD8+ T cell responses elicited by HA DNA-LNP, we next sought to determine whether these responses were durable at lower doses. We immunized mice with de-escalating doses of either HA DNA-LNP or mRNA-LNP and addressed antigenspecific T cell responses 14 days post immunization. Importantly, mice immunized with 0.2pg of HA DNA-LNP exhibited similar IFNy- secreting CD8+ T cell responses to 2pg of nucleoside- modified mRNA-LNP, demonstrating 10-fold dose sparing (FIG. 4G). Similar trends were observed with CD107a-expressing (FIG. 4H) and TNFa- secreting (FIG. 41) CD8+ T cell responses, where 0.2pg of HA DNA-LNP and 2pg of HA mRNA-LNP were functionally equivalent. HA DNA-LNP and HA mRNA-LNP elicited similar levels of IFNy- secreting (FIG. 9A), TNFa-secreting (FIG. 9B), and IL-2-secreting (FIG. 9C) CD4+ T cells at varying doses. Thus, these data demonstrate that HA DNA- LNP elicit robust CD8+ T cell responses that are durable at low doses.

[00416] SARS-CoV-2 spike-expressing DNA-LNP elicits GC and serum antibody titers

[00417] To extend these findings to an additional pathogen antigen, we generated ionizable LNP-formulated DNA and mRNA vaccines expressing full-length SARS-CoV-2 spike (41-43). Mice were immunized with 2pg of either pVAX DNA-LNP, spike DNA-LNP, spike mRNA-LNP, as well as licensed mRNA- LNP vaccine BNT162b2 and cellular responses were examined 14 days post immunization. In the DLNs, spike DNA-LNP elicited T follicular helper (Tfh) cell (FIG. 5A, FIG. 10A), GC B cell (FIG. 5B, FIG. 10B), and spike-specific GC B cell responses (FIG. 5C, FIG. 10C) comparable to that of nucleoside-modified spike mRNA-LNP in terms of frequency and cell number. Additionally, relative to BNT162b2, spike DNA-LNP immunized mice elicited a comparable spike-specific GC B cell responses. Thus, formulation of spike plasmid DNA in LNPs generates GC responses comparable to that of spike mRNA-LNP and a clinically validated BNT162b2.

[00418] To examine the induction of humoral responses in the periphery after immunization with spike DNA- LNP, mice were immunized with either control pVAX DNA-LNP, spike DNA-LNP, spike mRNA-LNP, or BNT162b2 and serum antibody responses were assessed 4 weeks post immunization. Spike DNA- LNP induced similar antibody responses to spike mRNA-LNP and BNT162b2 against matched spike RBD (FIG. 5D); RBD binding antibodies are associated with neutralization potency (44,45). Additionally, DNA-LNP elicited serum responses competitive to both spike mRNA-LNP and / or BNT162b2 against near wildtype or distant VOCs spike trimers, including D614G (FIG. 5E), Delta (FIG. 5F), and BA.2 (FIG. 5G). These data show spike DNA-LNP can elicit competitive antibody titers to spike mRNA- LNP against matched and distant SARS-CoV-2 spike antigens. To determine the functional potency of serum antibody titers, we assessed neutralization titers of serum antibody against wild-type SARS-CoV-2 pseudovirus. Spike DNA-LNP induces competitive but attenuated neutralization titers compared with spike mRNA-LNP and BNT162b2 against wildtype SARS- CoV-2 pseudovirus (FIG. 5H). Together, these data demonstrate that spike DNA-LNP can elicit functional serum antibody titers to SARS-CoV-2 similar to spike mRNA-LNP immunogens, including the licensed BNT162b2 vaccine, at the same dose.

[00419] Spike-expressing DNA-LNPs induce cytotoxic T cell responses

[00420] Next, we sought to determine whether plasmid DNA-LNP could elicit antigenspecific T cell responses in comparison with the two studied mRNA-LNPs. Mice were immunized as above, sacrificed 14 days later and splenocytes stimulated with peptides spanning the SARS-CoV-2 spike protein and T cell cytokine secretion was examined using flow cytometry. Spike DNA-LNP elicited robust and comparable IFNy-secreting (FIG. 6A), CD 107a- expressing (FIG. 6B), and TNFa-secreting (FIG. 6C) CD8+ T cell responses to mRNA-LNP and BNT162b2. In the context of CD4+ T cells, spike DNA-LNP elicited a superior IFNy-secreting CD4+ T cell response to that of spike mRNA-LNP and BNT162b2 (FIG. 6D). TNFa-secreting and IL-2-secreting CD4+ T cell responses between the three vaccines were comparable (FIG. 6E and FIG. 6F). Thus, these data demonstrate that DNA-LNP can elicit robust antigen-specific CD8+ and CD4+ T cell responses comparable or superior to mRNA-LNPs at the same dose. Table 1: In vitro biop lysical characterization of DNA-LNPs and mRNA-LNPs. LNPs Size (nm) (average) PDI (average) Zeta Potential (mV) (average) Encapsulation Efficiency % (average) C A09 HA DNA-LNPs (40:1) 74.69 0.145 -2.7 91% CA09 HA DNA-LNPs (20:1) 85.13 0.108 -3.33 89% CA09 HA DNA-LNPs (10:1) 122.63 0.154 -21.91 80.2% pVAX DNA-LNPs 77.92 0.145 -5.7 93.15 Spike DNA-LNPs 86.73 0.245 -0.76 88.2 Spike mRNA-LNPs 76.71 0.265 -1.4 91.5 CA09 HA DNA-LNPs 68.25 0.147 -4.6 90.4 CA09 HA mRNA-LNPs 68.72 0.205 -1.83 94.2 Table 2: Seq uences SEQ ID NO: Sequence Type Description 1 nucleotide SARS-CoV-2 RBD 2 amino acid SARS-CoV-2_RBD 3 nucleotide Flu_CA09_HA 4 amino acid Flu_CA09_HA 5 nucleic acid pVAX SARS-CoV-2_RBD (SEQ ID NO:1) ATGGACTGGACTTGGATTCTGTTCCTGGTCGCAGCAGCCACTCGGGTGCATAGCC GCGTGCAGCCCACTGAAAGCATTGTGAGATTCCCTAACATCACCAATCTGTGCCC ATTCGGCGAGGTGTTTAACGCCACACGGTTCGCCAGCGTGTACGCCTGGAACAGG AAGAGAATCTCCAATTGCGTGGCCGACTACTCTGTGCTGTATAATAGCGCCTCCTT CTCTACCTTTAAGTGCTACGGCGTGTCTCCCACCAAGCTGAACGACCTGTGCTTCA CAAACGTGTACGCCGACAGCTTTGTGATCAGGGGCGATGAGGTGAGACAGATCGC ACCAGGACAGACCGGCAAGATCGCAGACTACAACTATAAGCTGCCCGACGATTTC ACAGGCTGCGTGATCGCCTGGAATAGCAACAATCTGGATTCCAAAGTGGGCGGCA ACTACAATTATCTGTACAGGCTGTTCAGAAAGAGCAACCTGAAGCCCTTTGAGCG GGACATCTCTACCGAGATCTACCAGGCCGGCAGCACACCTTGCAACGGCGTGGAG GGCTTCAATTGTTACTTTCCACTGCAGTCTTATGGCTTCCAGCCCACAAACGGCGT GGGCTACCAGCCTTATCGCGTGGTGGTGCTGAGCTTTGAGCTGCTGCACGCACCA GCAACCGTGTGCGGACCTAAGAAGAGCACAAACCTGGTGAAGAATAAG (SEQ ID N0:2) MDWTWILFLVAAATRVHSRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRI SNC VAD YS VL YNS ASF STFKC YGVSPTKLNDLCFTNVYAD SF VIRGDEVRQIAPGQTG KIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQ AGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST NLVK NK Flu_CA09_HA (SEQ ID NO:3) ATGGACTGGACTTGGATTCTTTTTCTCGTGGCTGCAGCAACTAGGGTGCACAGCAT GAAGGCCATCCTGGTGGTGCTGCTGTACACCTTCGCCACAGCCAATGCTGACACC CTGTGCATCGGCTACCACGCCAACAACAGCACAGACACAGTGGACACAGTGCTGG AGAAGAACGTGACAGTGACCCACTCTGTCAACCTGCTGGAGGACAAGCACAACG GCAAGCTGTGTAAACTTCGAGGAGTTGCTCCTCTGCACCTGGGCAAATGTAACAT TGCTGGCTGGATTCTGGGCAACCCTGAATGTGAGAGCCTGAGCACAGCCAGCAGC TGGAGCTACATCGTGGAGACCCCTTCTTCTGATAATGGCACCTGCTACCCTGGAGA CTTCATCGACTATGAAGAGCTGAGAGAGCAGCTGTCCTCTGTTTCTTCTTTTGAAA GATTTGAAATCTTCCCCAAGACCAGCAGCTGGCCCAACCACGACTCTAACAAAGG AGTCACAGCTGCCTGTCCTCATGCTGGAGCCAAGTCCTTCTACAAGAACCTCATCT GGCTGGTGAAGAAGGGCAACAGCTACCCAAAGCTGTCCAAGTCCTACATCAACGA CAAGGGCAAGGAGGTGCTGGTGCTGTGGGGCATCCACCACCCTTCTACATCTGCT GACCAGCAGAGCCTGTACCAGAATGCTGATGCCTACGTCTTTGTGGGCAGCAGCA GATACAGCAAGAAGTTCAAGCCTGAGATCGCCATCAGACCTAAAGTTAGAGATCA AGAAGGACGCATGAACTACTACTGGACCCTGGTGGAGCCTGGAGACAAGATCAC CTTTGAGGCCACAGGAAACCTGGTGGTGCCAAGATATGCCTTCGCCATGGAGAGA AATGCTGGCAGCGGCATCATCATCTCTGACACACCTGTGCACGACTGCAACACCA CCTGCCAGACACCTAAAGGAGCCATCAACACCAGCCTGCCTTTCCAGAACATCCA CCCCATCACCATCGGCAAATGTCCTAAATACGTGAAGAGCACCAAGCTGCGGCTG GCCACAGGCCTGAGAAACATCCCTTCCATCCAGAGCAGAGGCCTGTTTGGAGCCA TCGCCGGCTTCATCGAGGGCGGCTGGACAGGCATGGTGGATGGCTGGTACGGCTA CCACCACCAGAATGAGCAGGGCAGCGGCTATGCTGCTGACCTGAAGAGCACCCA GAACGCCATCGATGAAATCACCAACAAGGTGAACAGCGTCATCGAGAAGATGAA CACCCAGTTCACAGCTGTGGGCAAGGAGTTCAACCACCTGGAGAAGAGAATTGAA AACCTGAACAAGAAGGTGGATGATGGCTTCCTGGACATCTGGACCTACAATGCTG AACTGCTGGTGCTGCTGGAAAACGAAAGAACACTGGACTACCACGACAGCAATGT GAAGAACCTGTACGAGAAGGTGAGAAGCCAGCTGAAGAACAACGCCAAGGAAAT TGGAAATGGCTGCTTTGAATTCTACCACAAGTGTGACAACACCTGCATGGAATCT GTGAAGAATGGCACCTACGACTACCCAAAGTACTCTGAAGAAGCCAAGCTGAAC AGAGAGGAAATCGATGGTGTGAAGCTGGAGAGCACCAGAATCTACCAGATCCTG GCCATCTACAGCACAGTGGCCAGCAGCCTGGTGCTGGTGGTGTCCCTGGGCGCCA TCTCCTTCTGGATGTGCAGCAACGGCAGCCTGCAGTGCAGAATCTGCATC (SEQIDN0:4) MDWTWILFLVAAATRVHSMKAILWLLYTFATANADTLCIGYHANNSTDTVDTVLE KNVTVTHSVNLLEDKHNGKLCKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYI VETPSSDNGTCYPGDFIDYEELREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPH AGAKSFYKNLIWLVKKGNSYPKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNA DAYVFVGSSRYSKKFKPEIAIRPKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPR YAFAMERNAGSGIIISDTPVHDCNTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKL RLATGLRNIPSIQSRGLFGAIAGFIEGGWTGMVDGWYGYHHQNEQGSGYAADLKSTQ NAIDEITNKVNSVIEKMNTQFTAVGKEFNHLEKRIENLNKKVDDGFLDIWTYNAELLV LLENERTLDYHDSNVKNLYEKVRSQLKNNAKEIGNGCFEFYHKCDNTCMESVKNGT YDYPKYSEE AKLNREEIDGVKLESTRIYQILAIYSTVAS SLVL WSLGAISF WMC SNGS LQCRICI pVAX (SEQ ID NO: 5) backbone sequence gactcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatag cccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataa tgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggactatttacggtaaactgcccacttggcagtaca tcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatg ggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagc ggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatg tcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactaga gaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagctggctagcgtttaaacttaagcttggtaccg agctcggatccactagtccagtgtggtggaattctgcagatatccagcacagtggcggccgctcgagtctagagggcccgtttaaacc cgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactc ccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggaca gcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctactgggcggttttatggacagc aagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttctcgccgccaag gatctgatggcgcaggggatcaagctctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggt tctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgt cagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaagacgaggcagcgcggctatcgt ggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgc cggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgat ccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggat gatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgagcatgcccgacggcgaggatctc gtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtg gcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgcttta cggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgaattattaacgcttacaatttcctgatgcg gtattttctccttacgcatctgtgcggtatttcacaccgcatacaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttattttt ctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatagcacgtgctaaaacttcatttttaatttaaa aggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaa gatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgcc ggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcg tgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccag cttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcg gacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtc ctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgc ggcctttttacggttcctgggcttttgctggccttttgctcacatgttctt

[00421] In some embodiments, a nucleic acid molecule comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to pVAX backbone or a functional fragment thereof and the nucleic acid sequence further comprises an expressible nucleic acid sequence within the multiple cloning site.

[00422] In some embodiments, a nucleic acid molecule comprises at least about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 1, 2, 3, or 4 or a functional fragment thereof and the nucleic acid sequence further comprises an expressible nucleic acid sequence within the multiple cloning site. References through Example 1: 1. Kutzler MA, Weiner DB. DNA vaccines: ready for prime time? Nature Reviews Genetics. 564 2008;9(10):776-88. 2. Tursi NJ, Xu Z, Kulp DW, Weiner DB. Gene-encoded nanoparticle vaccine platforms for in vivo assembly of multimeric antigen to promote adaptive immunity. Wiley Interdiscip Rev Nanomed Nanobiotechnol. 2023;15(4):el880. 3. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines — a new era in vaccinology. Nature Reviews Drug Discovery. 2018; 17(4):261-79. 4. Travieso T, Li J, Mahesh S, Mello JDFRE, Blasi M. The use of viral vectors in vaccine development, npj Vaccines. 2022;7(l):75. 5. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, et al. Efficacy and Safety of the mRNA-1273 SARS-CoV-2 Vaccine. New England Journal of Medicine. 2020;384(5):403-16. 6. Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine. New England Journal of Medicine. 2020;383(27):2603-15. 7. Hogan MJ, Pardi N. mRNA Vaccines in the COVID-19 Pandemic and Beyond. Annual Review of Medicine. 2022;73(l): 17-39. 8. Kariko K, Muramatsu H, Welsh FA, Ludwig J, Kato H, Akira S, et al. Incorporation of pseudouridine into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol Ther. 2008;16(ll):1833-40. 9. Kariko K, Buckstein M, Ni H, Weissman D. Suppression of RNARecognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity. 2005;23(2): 165-75. 10. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nature Reviews Materials. 2021;6(12): 1078-94. 11. Alameh M-G, Tombacz I, Bettini E, Lederer K, Ndeupen S, Sittplangkoon C, et al. Lipid nanoparticles enhance the efficacy of mRNA and protein subunit vaccines by inducing robust T follicular helper cell and humoral responses. Immunity. 2021;54(12):2877-92.e7. 12. Li C, Lee A, Grigoryan L, Arunachalam PS, Scott MKD, Trisal M, et al. Mechanisms of innate and adaptive immunity to the Pfizer-BioNTech BNT162b2 vaccine. Nature Immunology. 2022;23(4):543-55. 13. Knezevic I, Liu MA, Peden K, Zhou T, Kang H-N. Development of mRNA Vaccines: Scientific and Regulatory Issues. Vaccines. 2021;9(2):81. 14. Pardi N, Hogan MJ, Weissman D. Recent advances in mRNA vaccine technology. Current Opinion in Immunology. 2020;65:14-20. 15. Baiersdbrfer M, Boros G, Muramatsu H, Mahiny A, Vlatkovic I, Sahin U, et al. A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA. Molecular Therapy - Nucleic Acids. 2019;15:26-35. 16. Muramatsu H, Lam K, Bajusz C, Laczko D, Kariko K, Schreiner P, et al. Lyophilization provides long-term stability for a lipid nanoparticle-formulated, nucleoside- modified mRNA vaccine. Mol Ther. 2022;30(5): 1941-51. 17. Gary EN, Weiner DB. DNA vaccines: prime time is now. Current Opinion in Immunology. 2020;65:21-7. 18. Tebas P, Roberts CC, Muthumani K, Reuschel EL, Kudchodkar SB, Zaidi FI, et al. Safety and Immunogenicity of an Anti-Zika Virus DNA Vaccine. New England Journal of Medicine. 2017;385(12):e35. 19. Tebas P, Yang S, Boyer JD, Reuschel EL, Patel A, Christensen-Quick A, et al. Safety and 609 immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: A preliminary report of an open- 610 label, Phase 1 clinical trial. eClinicalMedicine. 2021 ;31. 20. Modjarrad K, Roberts CC, Mills KT, Castellano AR, Paolino K, Muthumani K, et al. Safety and immunogenicity of an anti-Middle East respiratory syndrome coronavirus DNA vaccine: a phase 1, open-label, single-arm, dose-escalation trial. The Lancet Infectious Diseases. 2019;19(9): 1013-22. 21. Trimble CL, Morrow MP, Kraynyak KA, Shen X, Dallas M, Yan J, et al. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2 / 3: a randomised, double-blind, 618 placebo-controlled phase 2b trial. The Lancet. 2015;386(10008):2078-88. 22. Xu Z, Patel A, Tursi NJ, Zhu X, Muthumani K, Kulp DW, et al. Harnessing Recent Advances in Synthetic DNA and Electroporation Technologies for Rapid Vaccine Development Against COVID-19 and Other Emerging Infectious Diseases. Frontiers in Medical Technology. 2020;2. 23. Khobragade A, Bhate S, Ramaiah V, Deshpande S, Giri K, Phophle H, et al. Efficacy, safety, and immunogenicity of the DNA SARS-CoV-2 vaccine (ZyCoV-D): the interim efficacy results of a phase 3, randomised, double-blind, placebo-controlled study in India. The Lancet. 2022;399(10332): 1313-21. 24. Flingai S, Czerwonko M, Goodman J, Kudchodkar S, Muthumani K, Weiner D. Synthetic DNA Vaccines: Improved Vaccine Potency by Electroporation and Co- Delivered Genetic Adjuvants. Frontiers in Immunology. 2013;4. 25. Hobernik D, Bros M. DNA Vaccines-How Far From Clinical Use? Int J Mol Sci. 2018;19(l 1). 26. De Rosa SC, Edupuganti S, Huang Y, Han X, ElizagaM, Swann E, et al. Robust antibody and cellular responses induced by DNA-only vaccination for HIV. JCI Insight. 2020;5(13). 27. Aggarwal C, Saba NF, Algazi A, Sukari A, Seiwert TY, Haigentz M, et al. Safety and Efficacy of MEDI0457 plus Durvalumab in Patients with Human Papillomavirus-Associated Recurrent / Metastatic Head and Neck Squamous Cell Carcinoma. Clin Cancer Res. 2023;29(3):560-70. 28. Mau T, Amin MR, Belafsky PC, Best SR, Friedman AD, Klein AM, et al. Interim Results of a Phase 1 / 2 Open-Label Study of INO-3107 for HPV-6 and / or HPV-11- Associated Recurrent Respiratory Papillomatosis. Fhe Laryngoscope. 2023; 133( 11):3087-93. 29. Cui L, Renzi S, Quagliarini E, Digiacomo L, Amenitsch H, Masuelli L, et al. Efficient Delivery of DNA Using Lipid Nanoparticles. Pharmaceutics. 2022;14(8). 30. Zhu Y, Shen R, Vuong I, Reynolds RA, Shears MJ, Yao Z-C, et al. Multi- step screening of DNA / lipid nanoparticles and co-delivery with siRNA to enhance and prolong gene expression. Nature Communications. 2022; 13(1):4282. 31. Algami A, Pilkington EH, Suys EJA, ALWassiti H, Pouton CW, Truong NP. In vivo delivery of plasmid DNA by lipid nanoparticles: the influence of ionizable cationic lipids on organ-selective gene expression. Biomaterials Science. 2022; 10(11):2940-52. 32. Ljungman P, Bermudez A, Logan AC, Kharfan-Dabaja MA, Chevallier P, Martino R, et al. A randomised, placebo-controlled phase 3 study to evaluate the efficacy and safety of ASP0113, a DNA-based CMV vaccine, in seropositive allogeneic haematopoietic cell transplant recipients.eClinicalMedicine. 2021;33. 33. Wang B, Ugen KE, Srikantan V, Agadjanyan MG, Dang K, Refaeli Y, et al. Gene inoculation generates immune responses against human immunodeficiency virus type 1. Proceedings of the National Academy of Sciences. 1993;90(9):4156-60. 34. Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Feigner PL, Dwarki VJ, et al. Heterologous Protection Against Influenza by Injection of DNA Encoding a Viral Protein. Science. 1993;259(5102):1745-9. 35. Zhang W, Pfeifle A, Lansdell C, Frahm G, Cecillon J, Tamming L, et al. The Expression Kinetics and Immunogenicity of Lipid Nanoparticles Delivering Plasmid DNA and mRNAinMice. Vaccines. 2023; 11(10): 1580. 36. Clogston JD, Patri AK. Zeta potential measurement. Methods Mol Biol. 2011;697:63-70. 37. Lederer K, Castano D, Gomez Atria D, Oguin TH, 3rd, Wang S, Manzoni TB, et al. SARS-CoV-2 mRNA Vaccines Foster Potent Antigen-Specific Germinal Center Responses Associated with Neutralizing Antibody Generation. Immunity. 2020;53(6):1281- 95.e5. 38. Porter KR, Raviprakash K. DNA Vaccine Delivery and Improved Immunogenicity. Curr Issues Mol Biol. 2017;22:129-38. 39. Korsholm KS, Petersen RV, Agger EM, Andersen P. T-helper 1 and T- helper 2 adjuvants induce distinct differences in the magnitude, quality and kinetics of the early inflammatory response at the site of injection. Immunology. 2010; 129(1):75-86. 40. Xu Z, Wise MC, Chokkalingam N, Walker S, Tello-Ruiz E, Elliott STC, et al. In Vivo Assembly of Nanoparticles Achieved through Synergy of Structure-Based Protein Engineering and Synthetic DNA Generates Enhanced Adaptive Immunity. Advanced Science. 2020;7(8): 1902802. 41. Gary EN, Warner BM, Parzych EM, Griffin BD, Zhu X, Tailor N, et al. A novel mouse AAV6 hACE2 transduction model of wild-type SARS-CoV-2 infection studied using synDNA immunogens. iScience. 2021;24(7): 102699. 42. Gary EN, Tursi NJ, Warner B, Parzych EM, Ali AR, Erase D, et al. Mucosal chemokine adjuvant enhances synDNA vaccine-mediated responses to SARS-CoV-2 and provides heterologous protection in vivo. Cell Reports Medicine. 2022;3(7): 100693. 43. Gary EN, Tursi NJ, Warner BM, Cuismano G, Connors J, Parzych EM, et al. Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge. Front Immunol. 2023; 14:1138609. 44. Xiaojie S, Yu L, Lei Y, Guang Y, Min Q. Neutralizing antibodies targeting SARS-CoV-2 spike protein. Stem Cell Res. 2020;50:102125. 45. Starr TN, Czudnochowski N, Liu Z, Zatta F, Park Y-J, Addetia A, et al. SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature. 2021;597(7874):97-102. 46. Gebre MS, Rauch S, Roth N, Gergen J, Yu J, Liu X, et al. mRNA vaccines induce rapid antibody responses in mice, npj Vaccines. 2022;7(l):88. 47. Liao H-C, Shen K-Y, Yang C-H, Chiu F-F, Chiang C-Y, Chai KM, et al. Lipid nanoparticle-encapsulated DNA vaccine robustly induce superior immune responses to the mRNA vaccine in Syrian hamsters. Molecular Therapy Methods & Clinical Development. 2024;32(l). 48. Gary DJ, Min J, Kim Y, Park K, Won YY. The effect of N / P ratio on the in vitro and in vivo interaction properties of PEGylated poly[2-(dimethylamino)ethyl methacryl ate] -based siRNA complexes. Macromol Biosci. 2013; 13(8): 1059-71. 49. Deal CE, Carfi A, Plante OJ. Advancements in mRNA Encoded Antibodies for Passive Immunotherapy. Vaccines (Basel). 2021;9(2). 50. Patel A, Bah MA, Weiner DB. In Vivo Delivery of Nucleic Acid-Encoded Monoclonal Antibodies. BioDrugs. 2020;34(3):273-93. 51. Laczko D, Hogan MJ, Toulmin SA, Hicks P, Lederer K, Gaudette BT, et al. A Single Immunization with Nucleoside-Modified mRNA Vaccines Elicits Strong Cellular and Humoral Immune Responses against SARS-CoV-2 in Mice. Immunity. 2020;53(4):724-32.e7. 52. Pardi N, Parkhouse K, Kirkpatrick E, McMahon M, Zost SJ, Mui BL, et al. Nucleoside-modified mRNA immunization elicits influenza virus hemagglutinin stalk- specific antibodies. Nat Commun. 2018;9(1):3361. Example 2: Plasmid DNA or mRNA formulated in lipid nanoparticles drive unique innate immune activation to promote adaptive immunity

[00423] Gene-vectored vaccines grew in importance over the past several years. However, understanding the differences between of lipid nanoparticle (LNP) formulations for delivering DNA and mRNA in particular has not been studied. Characterization of LNP-formulated DNA compared with mRNA could build upon current genetic delivery approaches. Here, we study a four-component ionizable LNP33 plasmid DNA formulation (DNA-LNPs) which we demonstrate induces potent innate and adaptive immunity at low doses with similar potency to mRNA-LNPs and adjuvanted protein. Using an influenza virus hemagglutininencoding construct (HA), we show that these DNA-LNPs drive potent inflammation dependent on the cGAS-STING-TBKl pathway but independent of TLR9. Priming with HA DNA-LNP demonstrated robust activation in migratory DC (mDC) subpopulations and significant upregulation of mDCs and neutrophils. Transcriptomics elucidated activation and upregulation of pro39 migration factors among multiple innate immune populations after priming with DNA-LNP. HA DNA40 LNP uniquely induced superior HA-specific CD8+ T cell responses relative to other platforms. HA DNA41 LNP additionally induced robust germinal center responses attenuated in frequency to mRNA-LNPs and adjuvanted protein, but with equivalent functional serum antibodies. Extending these findings to an additional pathogen antigen, SARS-CoV-2 spike-encoding DNA-LNP elicited protective efficacy comparable to spike mRNA-LNPs. Thus, this study identifies priming mechanisms and characterizes immune phenotypes after DNA-LNP immunization, suggesting additional avenues for vaccine development.

[00424] Vaccine approaches that can elicit both arms of adaptive immunity are valuable tools for combatting diverse pathogens as correlates of protection are frequently complex or unvalidated, exemplified with an emerging infectious disease such as severe acute respiratory syndrome coronavirus 2 (SARS55 CoV-2). Genetic vaccines, such as nucleic acid and viral-vectored, can elicit both humoral and cellular immunity (1-4) and can have unique production advantages.

[00425] Lipid nanoparticle-formulated nucleoside-modified mRNA vaccines (mRNA-LNPs) have demonstrated efficacy for the prophylaxis of COVID-19, with two initial product licenses for Modema’s SpikeVax (mRNA-1273) and Pfizer-BioNTech’s Comirnaty (BNT162b2) (5, 6). Immunization with mRNA-LNPs in animal models and humans is associated with robust immunogenicity (3, 7). Translation of mRNA has been reported to be generated 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 (1012). Some LNP formulations have also been reported to adjuvant recombinant protein vaccines (11).

[00426] IVT mRNA reactions have distinct synthetic requirements including template DNA, RNA polymerase, and ribonucleotides to form mRNA transcripts as well as a process to add a 5’ cap structure and polyA tail (13, 14). Modified ribonucleotides are commonly added rather than unmodified to reduce innate immune activation during immunization (8, 9). After transcription, IVT mRNA requires purification to remove byproducts of the reaction, such as double-stranded RNA species (15). Additionally, mRNA thermostability leads to cold chaindependent storage requirements. Recently, there have been advances for improving mRNA-LNP vaccine stability such as lyophilization (16). Overall, additional genetic vaccine tools with unique immunology remain important to evaluate.

[00427] DNA vaccines have demonstrated clinical safety and efficacy while also conferring advantages relative to some platforms including temperature stability and simple production (17-22). Recent advancements in DNA vaccine technology have led to clinical efficacy in two models of cancer therapy (23, 24). Naked DNA is poorly immunogenic as has been previously reported, and advancements in device delivery have improved in vivo immunogenicity and clinical study impact (1). 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 (25- 27). The direct encoding of gene adjuvant sequences (e.g., cytokines) to further enhance DNA vaccine-induced responses has increased potency in preclinical models and in the clinic (23, 28, 29). Additional tools for understanding and improving in vivo DNA vaccine immunogenicity remain important, considering the favorable manufacturing and stability aspects of plasmid DNA.

[00428] Lipid nanoparticle formulations of DNA have been studied, and formulations resulting in robust transfection efficiency in vivo have been reported (30-32), with recent progress in this area (33, 34). However, lipid-based formulation of plasmid DNA has demonstrated lower than optimal in vivo immunogenicity in the clinic (35). It has been hypothesized that DNA delivered in the cytoplasm is inefficiently transferred to the nucleus; early studies using naked DNA as vaccines suggest that expression from the transgene does occur without direct transfection into the nucleus (36, 37). Critically, LNP-encapsulated DNA could be of value as it retains the positive 96 product profile of DNA vaccines and device-free formulation would provide additional options for this platform in combination or separately from current delivery systems.

[00429] Here, we demonstrate that formulation of plasmid DNA into LNPs (DNA-LNPs) elicit robust immune responses with a unique phenotype relative to mRNA-LNPs and adjuvanted protein. We studied the immune response elicited from DNA-LNPs encoding two viral antigens, influenza virus A / California / 04 / 2009 hemagglutinin (CA09 HA) and SARS-CoV-2 spike protein. We observed that GC B cell responses were sensitive to formulation modifications while the T cell compartment remained intact. After down selecting on the most immunogenic formulation, we observed HA DNA-LNP induced robust inflammatory cytokine induction in local draining lymph nodes (DLNs) after immunization. Proinflammatory responses were dependent on the double stranded DNA (dsDNA) sensing pathway cGAS-STING-TBK1 but were independent of CpG-sensing toll-like receptor 9 (TLR9). Innate and adaptive populations showed robust activation 24 hours post immunization with HA DNA-LNP, particularly migratory dendritic cell (mDC) subpopulations in the draining lymph nodes (DLNs) and spleen. Uniquely, DNA-LNP immunization was associated with an upregulation in neutrophils at both proximal and distal immune sites. Single cell transcriptomics of early innate populations revealed a novel pro-activation and pro-migration profile among multiple innate immune subsets after priming with DNA-LNP. All immunization platforms studied led to robust upregulation of interferon (IFN) associated genes. We next studied the adaptive immune induction in further detail after immunization with CA09 HA-expressing DNA-LNP. HA DNA-LNP elicited a unique several fold enhancement in CD8+ T cell responses compared with both HA mRNA-LNP as well as adjuvanted protein. Analysis of the humoral compartment revealed slightly attenuated germinal center (GC) responses with HA DNA LNP immunization. However, HA DNA-LNP elicited robust serum antibody responses with comparable breadth and functionality to mRNA-LNP and adjuvanted protein. Extending to an additional pathogen antigen, spike-expressing DNA-LNP induced significant serological responses and were similar in protective efficacy to mRNA-LNP in a lethal SARS-CoV-2 challenge model. Together, these studies illustrate that LNP-formulated plasmid DNA exhibits a unique innate priming phenotype dependent on dsDNA sensing via cGAS-STING-TBKl. Further, DNA-LNP formulations exhibit dose-sparing humoral potency while additionally inducing superior CD8+ T cell responses, which provides an additional option for tailoring responses in varied immunization protocols. Results

[00430] Relationship between lipid components and DNA impacts immunogenicity

[00431] The association between lipid amine groups to nucleic acid backbone phosphates (N / P ratio) in LNP formulated nucleic acids can modulate immunogenicity. Plasmid is double stranded (having many more phosphate groups) and includes significantly longer sequences compared to mRNA expression cassettes. We examined whether different N / P ratios impact biophysical parameters and immunogenicity. N / P ratios for mRNA-LNPs have been well characterized, with a 6:1 N / P ratio utilized for licensed SARS-CoV-2 vaccines (38). We formulated CA09 HA-expressing plasmid DNA within LNPs (HA DNA-LNP) at a range of N / P ratios including 10.5, 5.3, and 2.6. Encapsulation efficiency was similar between 10.5 and 5.3 (91% and 89% respectively) but lower for 2.6 N / P ratio, approaching 80%. Additionally, 2.6 N / P ratio formulation particles were larger in size with a lower (more anionic) zeta potential (FIGS. 16A-16C). Nanoparticles with a zeta potential between -10 and 10 mV are considered neutral and associated with less cell wall destruction-associated toxicity seen with cationic particles (39). To examine how this relationship impacts immunogenicity, mice were immunized with HA DNA-LNPs at different N / P ratios or empty vector DNA-LNP (pVAX DNA-LNP) at a 10.5 N / P ratio. We used the antigen-specific GC and splenic T cell responses as an immunogenic readout 14 days post immunization. Interestingly, there was a significant attenuation in the frequency of total GC B cells (FIG. 11A-11B) with formulation at 5.3 and 2.6. We also observed a trend toward a decrease in HA-specific GC B cells with the 2.6 formulation (FIG. 11C). In the T cell compartment, we observed a similar activated T follicular helper (Tfh) cell response with formulations at all three N / P ratios (FIG. 11D). Additionally, in line with our Tfh cell data, antigen-specific effector CD4+ and CD8+ T cell responses appear unaffected with different lipid to DNA ratios. We do not observe a significant difference in interferon (IFN)y-secreting cells by enzyme-linked immunospot (ELISpot) assay (FIG. 1 IE) or cytokine-expressing CD8+ (FIGS. 16A-16I) or CD4+ T cells (FIGS. 16G-16I). Taken together, these data suggest that higher N / P ratios for plasmid DNA-LNPs lead to improved B cell responses within the GC, but both cytotoxic and helper T cell responses remain durable even at low N / P ratios.

[00432] DNA-LNPs induce cGAS-STING pathway-dependent inflammation

[00433] From these preliminary data, we sought to characterize the immune response in depth after immunization with plasmid HA DNA-LNP with lipids at a higher N / P ratio (10.5) in comparison with benchmark HA-encoding mRNA-LNP as well as protein in Addavax adjuvant, which is similar to MF59, the adjuvant in FDA-approved Fluad. First, we characterized innate immune sensing of DNA-LNPs in the DLNs through induction of local proinflammatory cytokine production. DLNs were harvested at 4 hours and 24 hours post immunization with HA DNA-LNP, HA mRNA-LNP, or adjuvanted HA protein and lysed to examine numerous pro-inflammatory cytokine mediators. Relative to a phosphate buffered saline (PBS)-only negative control, immunization with both DNA-LNP and mRNA-LNP at 4 and 24 hours led to robust upregulation of a number of different pro-inflammatory cytokines, notably IFNy and interleukin 6 (IL-6) (FIG. 11F-11G). Levels trended downward over time, which ameliorated responses at 24 hours relative to 4 hours. mRNA-LNP led to increased IFNy-induced protein 10 (IP-10) upregulation at 4 hours which was largely similar with DNA-LNP at 24 hours. Together, these data suggest that DNA-LNPs, along with mRNA-LNPs, are potent drivers of early inflammatory responses.

[00434] To gain a mechanistic understanding of how DNA-LNPs drive inflammatory responses, we treated naive splenocytes ex vivo with HA DNA-LNP with or without various chemical inhibitors of DNA sensing pathways and measured the production of both IFNa and IFNy. Treatment with DNA-LNP alone led to robust production of IFNa and IFNy, while plasmid DNA alone did not, suggesting that LNP-mediated delivery of plasmid DNA is driving innate immune inflammation (FIG. 11H-11I). However, in the presence of inhibitors to the double stranded DNA (dsDNA) sensing cGAS-STING-TBK1 pathway, we observe little to no secretion of either IFNa and IFNy. This is in contrast to CpG-sensing TLR9, which when inhibited does not impact inflammatory responses to either IFNa and IFNy. Together, these data suggest that sensing of dsDNA via cGAS-STING-TBKl (FIG. 11 J) is a driver of inflammatory responses to DNA-LNPs, while TLR9 sensing of CpG motifs is dispensable.

[00435] HA DNA-LNP induces robust early adaptive and innate immune activation

[00436] Considering the robust inflammatory responses we observed by HA DNA-LNP, we profiled early activation of both adaptive and innate immune populations that might occur during vaccine priming. Previous work has demonstrated that both mRNA-LNP vaccines (e.g., BNT162b2) and Addavax formulated protein elicit robust innate immune modulation in the draining lymph node (DLN), particularly mediated through myeloid activation (12, 40). We examined adaptive immune activation in both the DLNs and spleen of mice vaccinated with HA DNA-LNP, HA mRNA-LNP, or adjuvanted HA protein 24 hours post immunization. Using the early activation marker CD69, we observed robust upregulation of CD69 expression on both CD8+ T cells (FIG. 12A and 12B) and CD4+ T cells (FIGS. 17A and 17B) from mice immunized with both DNA-LNP and to a lesser extent mRNA-LNP, with approximately 7080% and 40-50% of T cells expressing CD69 in DNA-LNP and mRNA-LNP respectively. This was observed in both DLNs as well as in the spleen, with the greatest discrepancy observed in splenic T cells with a 3.5-4-fold increase in CD69+ expression with DNA-LNP immunization over mRNA-LNP. Profding innate natural killer (NK) cells demonstrated the same phenomenon, where we observed robust upregulation of CD69 expression in both DNA-LNP and mRNA-LNP immunization in the DLN, but only DNA-LNP immunization was observed to drive NK cell activation in the spleen (FIGS. 17C and 17D). Among NK cells, DNA-LNP immunization also uniquely led to secretion of IFNy in both DLNs as well as in the spleen (FIGS. 17C and 17D). Taken together, these findings indicate that DNA-LNP and mRNA-LNP immunization both lead to robust and early activation of T and NK cells at local sites, with DNA-LNP uniquely driving a distal activation phenotype.

[00437] Considering the T and NK cell activation profile we observed after DNA-LNP immunization, we next profiled innate immune populations more in depth using multiparameter flow cytometry 24 hours post immunization (FIG. 12E). We observed numerous immunization platform-specific changes in myeloid populations. There was a robust induction in total migratory dendritic cells (mDCs) in the DLN with DNA-LNP contrasting with a marked decline in these populations after mRNA-LNP or adjuvanted protein immunization (FIG. 12F). CD1 lb+ mDCs followed a similar trend, with a decline in mRNA-LNP and protein groups relative to DNA-LNP (FIG. 12G). There was a stark increase in CD103+ mDCs with DNA-LNP immunization relative to both control and the two other platforms (FIG. 12H). All platforms drove a decrease in the frequency of plasmacytoid DCs (pDCs) (FIG. 121). We also observed unique platform-specific changes in other myeloid populations. Immunization with DNA-LNP led to a striking increase in the frequency of neutrophils relative to other platforms or the control naive baseline (FIG. 2J). However, mRNA-LNP immunization uniquely drove an increase in the frequency of monocytes in DLNs (FIG. 12K). Focusing on the expression of activation marker CD86 by flow cytometry, we interrogated the activation profile among innate immune subsets. DNA-LNP immunization induced robust activation in all subsets, particularly in total mDC and both CDllb+ and CD103+ mDCs (FIG. 12L-12M). Relative to mDCs, we observed lower overall CD86 expression on neutrophils, pDCs, and monocytes, however DNA-LNP elicited significantly more CD86 expression on these populations. In the spleens of immunized animals, immunization with DNA-LNP, and to a lesser extend mRNA-LNP, led to downregulation in the frequency of total resident DCs (rDCs) (FIG. 18A), CD1 lb+ rDCs (FIG. 18B), and CD8a+ rDCs (FIG. 18C). However, the frequencies of mDCs in the spleen remained largely similar to naive, with a downregulation in total mDCs with protein immunization (FIG. 18D), downregulation in CDllb+ mDCs with mRNA-LNP immunization (FIG. 18E), and an upregulation in CD 103+ mDCs with both mRNA-LNP and protein immunization (FIG. 18F). This is in contrast to the DLN, with a striking increase in mDC populations after DNA-LNP immunization, indicating differential modulation of DC populations at proximal and distal sites. Similar to the DLN, we observed a robust upregulation in neutrophils after DNA-LNP immunization (FIG. 18G). However, in contrast to the DLN, we observed an upregulation in pDCs with mRNA-LNP immunization, which was unique among all treatment groups (FIG. 18H). In terms of activation profile, DNA-LNP immunization induced robust CD86 expression among rDC and mDC populations, despite decreases in total frequency (FIG. 181). In contrast, mRNA-LNP immunization led to a slight increase in CD86 expression in neutrophils and, along with DNA-LNP immunization, in pDCs. Taken together, these data suggest that DNA-LNP induces robust innate immune activation at proximal and distal sites and uniquely upregulates the frequency of migratory DC subpopulations and neutrophils 24 hours post immunization in the DLN.

[00438] Single cell transcriptomics highlights platform-specific modulation of innate immune populations

[00439] To further elucidate the mechanisms of innate immune modulation after vaccination with DNA-LNP, we performed single cell RNA sequencing (scRNAseq) on sorted DLNs 24 hours post immunization. We sorted CD45+CD19-TCRP- cells to enrich the nonlymphoid fraction for sequencing analysis. After quality control and clustering using shared nearest neighbors, we utilized uniform manifold approximation and projection (UMAP) for visualization of innate immune populations. We identified 5 clusters each corresponding to a different innate immune subset - NK cells, neutrophils, monocytes, pDCs, and conventional DCs (eDCs) (FIG. 13A). Sample wise distribution demonstrates platform specific modulation of populations, particularly in neutrophil, monocyte, and pDC clusters (FIG. 13B). To understand the activation signature after immunization with DNA-LNP, we examined the top differentially expressed genes within each cluster that were uniquely up or downregulated in DNA LNP relative to naive and the other vaccination platforms. Among NK cells, we observed downregulation and upregulation of C-C motif chemokine 5 (Cc / 5) and RUNX family transcription factor 3 (RunxS) respectively; Runx3 is important for NK cell activation and improved function within the tumor (FIG. 13C) (41, 42). Additionally, we observed tumor necrosis factor (TNF) receptor associated factor 1 (Trafl) upregulation in NK cells after DNA-LNP immunization, a gene downstream of TNF superfamily receptors induced by genes such as TNFa (43). In neutrophils, we observed upregulation of migration-associated genes such as chemokine Ccl4 and atypical chemokine receptor Ccrl2 (FIG. 13D) (44, 45). Additionally, we observed downregulation of Cd244a in neutrophils from DNA LNP immunized mice; CD244 expression is a functional identifier of myeloid-derived suppressor cells (46). Among monocytes, we observed unique upregulation of numerous chemokine or chemokine receptor genes associated with immune cell migration, such as Ccl4, Ccl2, Ccr5, and Cell2 (FIG. 13E). Both Ccl2 and Ccl4 - MCP-1 and MIP-ip, respectively - were upregulated at the protein level in the DLNs at 4 hours and 24 hours post immunization (FIG. 1 IF—11G). In pDCs, we observed upregulation of pro-inflammatory chemokine Ccl5 and alarmin S100a8, implicated in chemotaxis and activation of innate immune subsets (FIG. 13F). Among eDCs, we observed a strong activation signature with upregulation of Cd80 and Cd86, the latter of which mirroring our flow cytometry data (FIG. 13G). We also observed upregulation of Extl, a glucosyltransferase implicated in DC migration (47); these findings also reflect the robust upregulation in mDC populations observed by flow cytometry. We next examined genes that were differentially expressed among all active vaccination groups relative to the naive control. A consistent pro-inflammatory signature consistent with IFN signaling among NK cells, neutrophils, monocytes, and pDCs in multiple gene families including IFN-inducible proteins (IFI family), IFN-induced proteins with tetratricopeptide repeats (IFIT family), IFN-induced transmembrane proteins (IFITM family), IFN-stimulated genes (ISG family), and IFN-regulatory factors (IRF family) was observed (FIGS. 19A-19D). Active vaccination also induced NK cells upregulation of proinflammatory cytokine genes including Ifng and Gzmb. Among eDCs, all vaccine groups led to an upregulation in Statl and Cd274 expression; CD274 (PD-L1) is a negative regulator of T cell activation through PD-1-PD-L1 interactions, acting as an immune checkpoint during priming (FIG. 19E). Taken together, these data demonstrate that immunization with DNA-LNPs generate a unique pro-activation and pro-migration signature in numerous innate immune subsets, with all active vaccination modalities resulting in potent IFN-mediated inflammatory responses.

[00440] Influenza virus HA-expressing DNA-LNPs elicit GC responses and functional...

Claims

1. A composition comprising:(i) a nucleic acid molecule comprising from about 80% to about 100% DNA, the nucleic acid molecule further comprising a nucleic acid sequence encoding one or a plurality of therapeutic agents;(ii) SM-102 or a derivative thereof;(iii) a cholesterol molecule or a derivative thereof;(iv) l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof; and(v) DMG-PEG-2000 or a derivative thereof;wherein (ii), (iii), (iv) and (v) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively; and wherein (ii), (iii), (iv) and (v) form a lipid nanoparticle encapsulating the nucleic acid molecule.

2. The composition of claim 1 further comprising one or a plurality of nucleic acid molecules with a total mass amount of from about 0.1 micrograms to about 400 micrograms.

3. The composition of claim 1 further comprising a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms.

4. The composition of any of claims 1 through 3, wherein the lipid to DNA weight ratio of the lipid nanoparticle is about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1.

5. The composition of any of claims 1 through 4, wherein the nucleic acid molecule comprises cDNA, linear DNA, circular plasmid DNA, mini-circle DNA, rolling circleamplified DNA product, artificial chromosomes, replicating DNA or any combination thereof.

6. The composition of any of claims 1 through 5, wherein the lipid nanoparticle comprises a spheroid shape and comprises from about 50 to about 180 nanometers at its longest width dimension.

7. The composition of any of claims 1 through 6, wherein the lipid nanoparticlecomprises a spheroid shape and comprises a diameter from about 50 to about 180 nanometers at its longest width dimension.

8. The composition of claim 7 further comprising a population of lipid nanoparticles encapsulating one or a plurality of nucleic acid molecules, wherein the population of lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10,000 nucleic acid molecules.

9. The composition of any of claims 1 through 8, wherein the lipid nanoparticle has a zeta potential of from about -2 millivolts to about -22 millivolts.

10. The composition of claim 8, wherein the population of lipid nanoparticles have an average zeta potential of from about -0.5 mV to about -6 mV.

11. The composition of claim 8, wherein the population of lipid nanoparticles have an average diameter of from about 70 nanometers to about 77 nanometers.

12. The composition of any of claims 1 through 11, wherein the nucleic acid molecule is free of RNA.

13. The composition of any of claims 1 through 12, wherein the composition is free of RNA.

14. The composition of any of claims 1 through 13, wherein the nucleic acid sequence encodes an antigenic determinant or a functional variant thereof.

15. The composition of claim 14, wherein the antigenic determinant comprises from about 8 to about 40 amino acids and is a pathogen antigen.

16. The composition of claim 15, wherein the antigenic determinant comprises a viral antigen.

17. The composition of claim 16, wherein the viral antigen is an influenza HA antigen or SARS-CoV-2 antigen.

18. The composition of claim 16, wherein the viral antigen comprises SEQ ID NO:2 or SEQ ID NO:4 or functional variants thereof comprising at least about 70% sequence identity to SEQ ID NO:2 or SEQ ID NO:4.

19. The composition of any of claims 1 through 18, wherein the nucleic acid molecule further comprises a second nucleotide sequence encoding a co-stimulatory molecule, a cytokine or a chemokine operably linked to a regulatory sequence.

20. The composition of any of claims 1 through 14, wherein the nucleic acid sequence encodes an antibody or antibody fragment thereof.

21. The composition of any of claims 1 through 20, wherein lipid nanoparticle comprises a targeting domain, at least a portion of which is positioned outside on the surface of the lipid nanoparticle.

22. The composition of claim 21, wherein the targeting domain comprises a binding molecule specific for binding to an antigen presenting cell.

23. The composition of claim 22, wherein the binding molecule is chosen from: CD 11c, BDCA-2, BDCA-3, BDCA-4, CD la, CDlc / BDCA-1, SIRP alpha / CD172a, CD1 Ib / Integrin alpha M, FLT3, CD40, CD80, and CD86, or functional variants thereof.

24. The composition of any of claims 1 through 23, wherein the composition is free of a molecule of Formula I:or a derivative thereof.

25. A pharmaceutical composition comprising: (i) a therapeutically effective amount of a composition of any of claims 1 through 24; and (ii) a pharmaceutically acceptable carrier.

26. The pharmaceutical composition of claim 25, wherein the pharmaceutical composition is a liquid dosage form and the nanoparticles are suspended in IX phosphate buffered saline of a pH of about 6.

27. A method of delivering a nucleic acid molecule into a cell comprising administering to a subject in need thereof a therapeutically effectively amount of a composition of any one of claims 1 through 24 or the pharmaceutical composition of any of claims 25 or 26.

28. The method of claim 27, wherein the DNA molecule comprises cDNA, linear DNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, artificial chromosomes, replicating DNA or any combination thereof.

29. The method of claim 27, wherein the nucleic acid sequence encodes a therapeutic agent.

30. The method of claim 29, wherein the therapeutic agent comprises one or a combination of two or more of: a viral antigen, a bacterial antigen, a fungal antigen, a parasitic antigen, an influenza antigen, a tumor-associated antigen, and a tumor-specific antigen.

31. A method of preventing or treating a disease or disorder in a subj ect in need thereof comprising administering the subject a therapeutically effectively amount of at least one composition of any one of claims 1 through 24 or the pharmaceutical composition of any of claims 25 or 26.

32. The method of claim 31, wherein the composition thereof delivers a DNA molecule encoding a therapeutic agent to the nucleus of a target cell of interest.

33. The method of claim 31, wherein the disease or disorder is a viral infection.

34. The method of claim 33, wherein the viral infection is influenza infection or SARS-CoV-2 infection.

35. A method of in vivo delivery of DNA molecules comprising administering to a subject in need thereof a therapeutically effectively amount of a composition of any one of claims 1 through 24 or the pharmaceutical composition of any of claims 25 or 2636. The method of claim 35, wherein the DNA molecule comprises cDNA, linearDNA molecules, circular plasmids or expression vectors, mini-circle DNA, rolling circle amplified DNA product, artificial chromosomes, replicating DNA or any combination thereof.

37. A method of vaccinating a subject in need thereof comprising administering to a subject in need thereof a composition of any of claims 1 through 22.

38. A method of inducing an antigen-specific immune response in a subject in need thereof comprising administering to a subject in need thereof with a composition of any of claims 1 through 22 , wherein the one or plurality of therapeutic agents comprises an antigen or antigenic determinant from a pathogen or an antigen associated with a hyperproliferative disorder.

39. A kit comprising the composition of any one of claims 1 through 24 or the pharmaceutical composition of any of claims 25 or 26.

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