Methods of making lipid nanoparticles and modulating the immune system using the same

Abstract

The disclosure relates to methods of manufacturing lipid nanoparticles comprising DNA for administration of gene therapy vectors or inducing antigen-specific immune responses in a subject by allowing the lipid nanoparticles to self-assemble under a fluid flow rate from about 1 mL per minute to about 10 mL per minute.

Description

ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONMETHODS OF MAKING LIPID NANOPARTICLES AND MODULATING THE IMMUNE SYSTEM USING THE SAMECROSS REFRENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 728,671, which was filed December 5, 2024, entitled “Methods of Making Lipid Nanoparticles and Modulating the Immune System Using the Samef and is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

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

[0003] 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.

[0004] Lipid nanoparticle-formulated nucleoside-modified mRNA vaccines (mRNA- LNPs) have demonstrated efficacy for the prophylaxis of SARS-CoV-2, with two initial product licenses from Moderna (mRNA-1273 / SpikeVax) and Pfizer-BioNTech (BNT162b2 / Comimaty) (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).ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0005] 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).

[0006] 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.

[0007] 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.

[0008] 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- BioNT ech 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

[0001] In some embodiments, the disclosure relates to a method of making a lipid nanoparticle. In some embodiments, the method comprises exposing a composition comprising aATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION lipid mixture and a deoxyribonucleic acid (DNA) molecule in a fluid circuit under a fluid flow rate of at least about 5 mb per minute. In some embodiments, the method further comprises allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the lipid mixture comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof.

[0002] In some embodiments, the composition further comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; and (iii) polyethylene glycol or a derivative thereof. In some embodiments, (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the nucleic acid molecule further comprises a nucleic acid sequence encoding one or a plurality of therapeutic agents. In some embodiments, the lipid nanoparticle further comprises a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms. In some embodiments, the lipid to DNA weight ratio of the lipid nanoparticle is about 40 to about 1; or from about 20 to about 1; or from about 10 to about 1. In some embodiments, the polyethylene glycol is DMG-PEG-2000 or a derivative thereof. In some embodiments, the DNA molecule is a cDNA molecule, a linear DNA molecule, a circular plasmid DNA molecule, a mini-circle DNA molecule, a rolling circle amplified DNA product, an artificial chromosome, a replicating DNA or any combination thereof. In some embodiments, the lipid nanoparticle comprises a spheroid shape or semi-spheroid shape. In some embodiments, the lipid nanoparticle comprises from about 50 to about 180 nanometers at its longest width dimension after spontaneous formation.

[0003] In some embodiments, the method further comprises repeating the step of exposing such that a population of lipid nanoparticles is manufactured and the lipid nanoparticles encapsulate one or a plurality of DNA molecules; and the population of lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10,000 nucleic acid molecules.

[0004] In some embodiments, the lipid nanoparticle or a population of lipid nanoparticles has a zeta potential of from about -2 millivolts to about -22 millivolts. In some embodiments, the population of lipid nanoparticles have an average zeta potential of from about -0.5 mV to about - 6 mV. In some embodiments, the population of lipid nanoparticles have an average diameter from about 70 nanometers to about 77 nanometers. In some embodiments, the composition and the lipid nanoparticle are free of RNA.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0005] In some embodiments, the method further comprises a step of mixing the lipid, the cholesterol, the polyethylene glycol and the SM-102 in an aqueous solution prior to exposing the lipid mixture to the DNA molecule.

[0006] In some embodiments, the step of exposing is performed within a fluid circuit with the flow rate in a lateral flow of solution under room temperature. In some embodiments, the step of exposing is performed on a system comprising a first reservoir, a second reservoir; and a pump. In some embodiments, each of the first and second reservoirs connected to a fluid circuit and the pump configured to create fluid flow in the fluid circuit. In some embodiments, the fluid circuit comprises a first set of conduits connecting the first reservoir and the second reservoir to a reaction region. In some embodiments, the system further comprises a processor, controller and computer program product. In some embodiments, the method is a computer-implemented method and the computer-program product comprises computer-executable instructions for: (a) initiating fluid flow from the first and second reservoirs; and (b) setting a fluid flow rate in the fluid circuit. In some embodiments, the computer program product comprises computer-executable instructions for (c) calculating the fluid flow rate in the fluid circuit.

[0007] In some embodiments, the fluid flow rate during the exposing step is from about 5 mL per minute to about 200 mL per minute. In some embodiments, the fluid flow rate during the exposing step is from about 5 mL per minute to about 100 mL per minute. In some embodiments, the fluid flow rate during the exposing step is from about 5 mL per minute to about 500 mL per minute. In some embodiments, the fluid flow rate is from about 5 mL per minute to about 20 mL per minute.

[0008] In some embodiments, the disclosure relates to a computer-implemented method of manufacturing a population of lipid nanoparticles in a system. In some embodiments, the system comprises a first reservoir, a second reservoir; and a pump. In some embodiments, each of the first and second reservoirs are connected to a fluid circuit and the pump is configured to create fluid flow in the fluid circuit. In some embodiments, the method comprises: (a) initiating fluid flow from the first reservoir into the fluid circuit; (b) initiating fluid flow from the second reservoir into the fluid circuit; and (c) exposing a composition comprising a lipid mixture and a deoxyribonucleic acid (DNA) molecule in a fluid circuit under a flow rate of from about 5 mL per minute to about 20 mL per minute. In some embodiments, the method further comprises allowing a time period sufficient for the lipids to spontaneously form a population of lipid nanoparticles and encapsulateATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION the DNA molecule. In some embodiments, the lipid mixture comprises 1,2-distearoyl-sn-glycero- 3 -phosphocholine (DSPC) or a derivative thereof. In some embodiments, the first reservoir comprises the lipid mixture and the second reservoir comprises an aqueous solution comprising the DNA molecule. In some embodiments, the lipid mixture further comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; and (iii) polyethylene glycol or a derivative thereof. In some embodiments, (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

[0009] In some embodiments, the lipid nanoparticle further comprises a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms. In some embodiments, the lipid to DNA weight ratio of the lipid nanoparticle is about 40 to about 1 ; or from about 20 to about 1 ; or from about 10 to about 1. In some embodiments, the polyethylene glycol is DMG-PEG-2000 or a derivative thereof.

[0010] In some embodiments, the disclosure relates to a method of modulating an immune response in a subject in need thereof. In some embodiments, the method comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 40 to about 1 if enhancing an immune response in the subject; or (b) administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio lower than about 40 to about 1 if reducing an immune response in the subject.

[0011] In some embodiments, the DNA comprises a nucleotide sequence encoding one or more therapeutic agents.

[0012] In some embodiments, the disclosure relates to a method of delivering a therapeutic agent to a subject by inducing the STING pathway independent of and / or free of stimulating TLR9. In some embodiments, the method comprises administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio from about 6: 1 to about 20: 1. In some embodiments, the average N / P ratio of the population of lipid nanoparticles is from about 4 to about 10.

[0013] In some embodiments, the disclosure relates to a method of reducing an antigenspecific immune response to a gene therapy vector in a subject in need thereof. In some embodiments, the method comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein theATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION lipid nanoparticle have a lipid to DNA weight ratio lower than about 20 to about 1. In some embodiments, the lipid nanoparticles are free of RNA encoding a therapeutic protein. In some embodiments, the lipid nanoparticles are free of RNA and the method is free of TLR9 stimulation in the subject. In some embodiments, the method further comprises manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 5 mL per minute to about 20 mL per minute. In some embodiments, the method further comprises allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the lipid mixture comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the composition further comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; and (iii) polyethylene glycol or a derivative thereof. In some embodiments, (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

[0014] In some embodiments, the disclosure relates to a method of selectively loading lipid nanoparticles with a mass amount of DNA. In some embodiments, the method comprises: (a) choosing a desired N / P ratio of the lipid nanoparticle; and (b) adjusting flow rate in a fluid circuit to arrive at a substantially homogeneous population of lipid nanoparticles with the desired N / P ratio. In some embodiments, if the desired N / P ratio is from about 1 to about 8, setting the flow rate of at least about 5 mL per minute. In some embodiments, the method further comprises (c) exposing a composition comprising a lipid mixture and a DNA molecule in the fluid circuit. In some embodiments, the method further comprises (d) allowing a time period sufficient for the lipids to spontaneously form a population of lipid nanoparticles and encapsulate the DNA molecule. In some embodiments, the lipid mixture comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the lipid mixture further comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; and (iii) polyethylene glycol or a derivative thereof. In some embodiments, (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, no less than 80% of the lipid nanoparticles in the population of lipid nanoparticles further comprise a plurality of DNA molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms. In some embodiments, the lipid to DNA weight ratio of the lipid nanoparticle is about 40 to about 1; or from about 20 to about 1;ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION or from about 10 to about 1 . In some embodiments, the polyethylene glycol is DMG-PEG-2000 or a derivative thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] 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.

[0010] FIGS. 1A through 1J depict how the flow rate of particle formation impacts LNP size at lower N / P ratios. The N / P ratio is the ratio of lipid amine groups to nucleic acid backbone phosphates. DNA-LNPs at N / P ratios ranging from 6 to 15 were formulated at varying flow rates. (FIG. 1 A) N / P = 6 (FIG. IB) N / P = 10.5 (FIG. 1C) N / P = 12 (FIG. ID) N / P = 15. FIGS. IE Proportionally, DNA-LNPs made at N / P 6 have at most 20% of particles loaded with DNA as determined with NanoFCM (FIG. IE). The majority of loaded LNPs had two copies of DNA (FIG. IF). DNA-LNPs made at N / P 10.5 had a larger fraction (>30%) of LNPs loaded with DNA at a flow rate of 3 and 6 mL per minute with a similar copy number profile (FIGS. 1G and 1H). DNA- LNPs made at N / P 15 and a flow rate of 6 ml per minute had greater than 40% of particles loaded with DNA, the majority containing 2 copies (FIGS. II and 1 J).

[0011] FIG. 2 depicts how the flow rate of particle formation impacts LNP size at lower N / P ratios. DNA-LNPs at N / P ratios ranging from 6 to 15 were formulated at varying flow rates.

[0012] FIGS. 3A through 3F depict data demonstrating that Spike DNA-LNP induces potent T cell responses. FIG. 3 A 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. 3B) CD107a+ CD8+ T cells shown as in (FIG. 3A). (FIG. 3C) TNFa+ CD8+ T cells shown as in (FIG. 3A). (FIG. 3D) 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. 3E) TNFa+ CD4+ T cells shown as in (FIG. 3D). (FIG. 3F) IL-2+ CD4+ T cellsATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION shown as in (FIG. 3D). 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.

[0013] FIGS. 4A and 4B depict data demonstrating that mouse muscle cells can be effectively transfected with lipid-formulated plasmid DNA. (FIG. 4A-FIG. 4B) C2C12 cells (mouse myoblast) were transfected via lipofectamine with plasmid DNA expressing GFP (FIG. 4A) 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.

[0014] FIGS. 5 A and 5B depict data demonstrating that the longevity of antibody titers in mice immunized with HA DNA-LNP. Mice were immunized with 2pg of HA DNA-LNP and serum titers were followed longitudinally, reported as raw OD (FIG. 5A) or endpoint titer calculated against naive animals (FIG. 5B). Dots represent individual animals; n=15 animals per group. Plots show geometric mean with geometric SD.

[0015] FIGS. 6A through 6C depict data demonstrating the dosing of DNA-LNP and mRNA-LNP elicits similar CD4+ T cell responses. Frequency of activated CD4+ T cells expressing IFNv (FIG. 6A), TNFa (FIG. 6B), or IL-2 (FIG. 6C) 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.

[0016] FIGS. 7A through 7C depict data demonstrating the absolute numbers of Tfh and GC B cells after immunization with Spike-expressing DNA or mRNA-LNPs. Mice were immunized as in FIG. 1. Bar plots show quantification of absolute numbers of Tfh (FIG. 7A), total GC B (FIG. 7B), and spike-specific GC B (FIG. 7B) 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.

[0017] FIGS. 8 A through 8 J illustrate initial immune characterization of HA DNA-LNP formulations and immunogenicity. FIG. 8A: 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. 8B : Bar plots quantifying frequency of GC B cells at 14 days post immunizationATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION in the DLNs. Frequency expressed as a percentage of CD19+ B cells. Pre-gated on live CD4- CD19+ FIG. 8C: 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. 8D: Frequency of activated Tfh cells expressed as a percentage of CD44+ CD4+ T cells. Pre-gated on live CD19- CD4+. FIG. 8E: IFNy ELISpot of splenocytes 14 days post immunization. FIGS. 8F and 11G: Fold change induction of cytokines in DLNs at 4 hours (FIG. 8F) and 24 hours (FIG. 8G) post immunization quantified using Luminex. FIGS. 8H and 81: ELISpot assay measuring IFNa (FIG. 8H) and IFNy (FIG. 81) 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. 8J: 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. 8H and 81); data pooled from two independent experiments (FIGS. 8A-8E, 8H, and 81) or from one independent experiment (FIGS. 8F and 8G). 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.

[0018] Fig. 9 illustrates that DNA-LNP does not induce TLR9 activation. HEK-Blue mouse TLR9 expressing cells were cultured with DNA-LNPs or stimulatory CpG 1826 at varying concentrations or inhibitory CpG 2088 and TLR9 activation was measured 20 hours post culture.DETAILED DESCRIPTION

[0019] 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.

[0020] 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0021] Definitions

[0022] 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.

[0023] As used herein, each of the following terms has the meaning associated with it in this section.

[0024] 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.

[0025] “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.

[0026] “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 -Cl 2 alkyl), one to eight carbon atoms (C1-C8 alkyl) or one to six carbon atoms (C1-C6 alkyl) and which is attached to the rest of the molecule by a single bond, e.g., methyl, ethyl, n propyl, 1-methylethyl (iso propyl), n butyl, n pentyl, 1,1 dimethylethyl (t butyl), 3 methylhexyl, 2 methylhexyl, ethenyl, prop 1 enyl, but-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.

[0027] 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 substituentsATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION selected from halogen, -OH, alkoxy, -NH2, tri fluoromethyl, -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.

[0028] “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 (C1-C15 alkylene), one to twelve carbon atoms (Cl -Cl 2 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.

[0029] “Cycloalkyl” or “carbocyclic ring” refers to a stable non aromatic monocyclic or polycyclic hydrocarbon radical consisting solely of carbon and hydrogen atoms, which may include fused or bridged ring systems, having from three to fifteen carbon atoms, preferably having from three to ten carbon atoms, and which is saturated or unsaturated and attached to the rest of the molecule by a single bond. Monocyclic radicals include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic radicals include, for example, adamantyl, norbornyl, decalinyl, 7,7 dimethyl bicyclo[2.2.1]heptanyl, and the like.

[0030] 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.

[0031] 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 quatemized. The heteroatom(s) may be placed at anyATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0032] “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, 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 quatemized; 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, 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.

[0033] 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 K (pi) electrons, where n is an integer.

[0034] 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[1] 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 areATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0035] 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.

[0036] 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-di oxane, homopiperazine, homopiperidine, 1,3-dioxepane, 4,7-dihydro-l,3-dioxepin and hexamethyleneoxide.

[0037] 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 and1.3.4-oxadiazolyl.

[0038] 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,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION1,5-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.

[0039] The aforementioned listing of heterocyclyl and heteroaryl moieties is intended to be representative and not limiting.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 C1-C12 alkyl or cycloalkyl); carboxyl groups (-OC(=O)Ra or -C(=O)ORa, where Ra is H, C1-C12 alkyl or cycloalkyl); amine groups (-NRaRb, where Ra and Rb are each independently H, Cl -Cl 2 alkyl or cycloalkyl); Cl- C12 alkyl groups; and cycloalkyl groups. In some embodiments the substituent is a C1-C12 alkylATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION group. In other embodiments, the substituent is a cycloalkyl group. Tn 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] “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 isATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION“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.

[0049] 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.

[0050] 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.

[0051] The term “DNA” as used herein is defined as deoxyribonucleic acid.

[0052] The term “RNA” as used herein is defined as ribonucleic acid.

[0053] “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.

[0054] “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 orATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0055] “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.

[0056] 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.

[0057] “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.

[0058] 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.

[0059] 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).

[0060] 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 mayATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION include introns. . In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translation by translational machinery in a cell.

[0061] 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.

[0062] 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.

[0063] 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).

[0064] 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0065] The term “recombinant polypeptide” as used herein is defined as a polypeptide produced by using recombinant DNA or RNA methods.

[0066] The term “recombinant DNA” as used herein is defined as DNA produced by joining pieces of DNA from different sources.

[0067] The term “recombinant RNA” as used herein is defined as RNA produced by joining pieces of RNA from different sources.

[0068] 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.

[0069] 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.

[0070] As used herein, the term “identical” refers to two or more sequences or subsequences which are the same.

[0071] 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 ofATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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 negative-scoring residue alignments; or 3) the end of either sequence is reached. The Blast algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The Blast program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff et al., Proc. Natl. Acad. Sci. USA, 1992, 89, 10915-10919, which is incorporated herein by reference in its entirety) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands. The BLAST algorithm (Karlin et al., Proc. Natl. Acad. Sci. USA, 1993, 90, 5873- 5787, which is incorporated herein by reference in its entirety) and Gapped BLAST perform a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication ofATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0072] 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.

[0073] “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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0074] 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.

[0075] 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.

[0076] 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 encodeATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0077] 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.

[0078] 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.

[0079] As used herein, the term “N / P ratio” means the ratio of lipid amine groups to nucleic acid backbone phosphates.

[0080] As used herein, “antigen” or “antigenic determinant” refers to a target molecule or portion thereof to which an immune protein; e.g., a T-cell receptor, an antibody, or functional fragments thereof, binds, or to a particular epitope on the target molecule or portion thereof to which an the immune protein binds.Compositions and Systems

[0081] 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.

[0082] 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.

[0083] 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 80ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0084] 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.

[0085] 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.

[0086] In various embodiments, the lipids or the LNP of the present disclosure are substantially non-toxic.

[0087] 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 orATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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%.

[0088] 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.

[0089] 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION26(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- di oleyloxy )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.).

[0090] 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- dilinol ey oxy-3 -(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoley oxy-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.Cl), 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).

[0091] In some embodiments, the LNP comprises a PEG derivative chosen from the following:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0135] 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, theATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONLNP 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%.

[0136] 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.

[0137] 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%.

[0138] 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 LNPATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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 concentrationATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION of about 95.5 mol%. Tn 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%.

[0139] 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, stearoyl oleoylphosphatidylcholine (SOPC) or a derivative thereof, l-stearioyl-2-oleoyl- phosphatidyethanol amine (SOPE) or a derivative thereof, N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethyl ammonium chloride (DOTAP) or a derivative thereof, or any combination thereof.

[0140] 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%.

[0141] 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.

[0142] In some embodiments, the cholesterol lipid is cholesterol or a derivative thereof.

[0143] 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%. InATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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%.

[0144] 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

[0145] 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%.

[0146] 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.

[0147] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (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-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearioyl-2-oleoyl-phosphatidy ethanol amine (SOPE), stearoyl oleoylphosphatidylcholine (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).

[0148] In some embodiments, the composition comprises a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0149] In some embodiments, the LNP comprises one or more steroid. A “steroid” is a compound comprising the following carbon skeleton:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0150] In some embodiments, the steroid or steroid analogue is cholesterol.

[0151] 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, diacylphosphatidyl serine, diacylphosphatidic acid, N- dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines, N- glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0152] 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 l-(monom ethoxy-poly ethyleneglycol)- 2,3-dimyristoylglycerol (PEG-s- DMG) and the like.

[0153] 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 glycol-lipid is PEG-c-DOMG). In other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as 1- (monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4- 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-ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate.

[0154] 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%.

[0155] 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.

[0156] 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).

[0157] 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 someATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION embodiments n is i or 2, and Ri comprises a first and a second non-contiguous alkyl or heteroalkyl chains covalently bound to a COO, wherein the first alkyl chain is from about 3 to about 5 contiguously bound carbon atoms in length and the second alkyl chain is from about 2 to about 14 carbon atoms in length. In some embodiments, n is 1, 2 or 3, and Ri is a C12 to C28 monoglyceride, alkenyl, alkyl, aryl, or aralkyl.

[0158] 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:

[0159] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0160] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0161] 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.

[0162] 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,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP.

[0163] LNP Compositions

[0164] 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.

[0165] 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.

[0166] 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%ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0167] 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONIn 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.

[0168] 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 aboutATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION-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.

[0169] 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 fromATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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.

[0170] In some embodiments, the composition is free of a molecule of Formula I:or a derivative thereof.

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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%.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONIn 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%.

[0175] 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%.

[0176] 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 fractionATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION is about 1.5%. In some embodiments, the fraction is about 2%. Tn 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%.

[0177] 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%.

[0178] 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0179] 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.

[0180] 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 tumorspecific antigen, or any combination thereof. In some embodiments, the invention includes a nucleic acid molecule encoding an adjuvant.

[0181] 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, minicircle 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.

[0182] 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.

[0183] 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.

[0184] 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 someATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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%.

[0185] In some embodiments of a composition herein, the LNP therein comprises an N / P ratio of about 0.5, about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is 0.5, 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.0, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from about 0.5 to about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2 to about 2.5, about 2.6, about 3, about3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 4 to about4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 5.3 to about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION about 9.5, about 10, about 10.5, about 11, about 11 .5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 7 to about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 9 to about 9.5, about 10, about10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 15 to about 15.5, about 16, about16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from 0.5 to 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2 to 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2.6 to 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or20. In some embodiments, the N / P ratio in the LNP is from 4 to 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 5.3 to 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 7 to 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 9 to 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 10.5 to 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 15 to 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION19.5, or 20. Tn some embodiments, the N / P ratio in the LNP is about 2.6, about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 5.3, about10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 5.3 to about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is about 2.6 to about 5.3. In some embodiments, the N / P ratio in the LNP is about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 15. In some embodiments, the N / P ratio in the LNP is 2.6, 5.3,10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 2.6 to 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 5.3 to 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 10.5 to 15. In some embodiments, the N / P ratio in the LNP is 2.6 to 5.3. In some embodiments, the N / P ratio in the LNP is 10.5 to 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than 15.

[0186] The disclosure also provides for a system within which one or a population of LNPs are formed. In some embodiments, the system comprises: an open or closed fluid circuit in fluid communication with a first reservoir, a second reservoir, the first and second reservoirs are removable at a first and a second inlet, respectively. In some embodiments, the fluid circuit comprises a pump operably linked to the first and second reservoir via one or a plurality of lengths of tubing or one or a plurality of conduits, and capable of creating fluid flow from the first and second reservoirs through a flow cell in a reaction region of the fluid circuit. In some embodiments, the reaction region is a point within the fluid circuit where branched conduits from the first andATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION second reservoirs meet to form a single cavity or space, such that fluid in the fluid circuit from the first and second reservoirs are mixed. In some embodiments, methods of forming the LNPs include use of the aforementioned system and the disclosed steps of exposing the lipid mixture and the DNA molecule are performed in the reaction region; wherein the first reservoir comprises an aqueous solution comprising the DNA molecule, and wherein the second reservoirs comprises an aqueous solution comprising the lipid mixture. Methods of the disclosure include operating one or two pumps operably linked to the first and second reservoirs, such that operation of the one or two pumps draws fluid from the first and second reservoirs into the fluid circuit through the one or plurality of conduits and into the reaction region.

[0187] In some embodiments, the fluid circuit comprises an outlet in fluid communication with the reaction region, such that fluid flow in the fluid circuit originates from the first and / or second reservoirs, passes through a first set of conduits, into the reaction region and through the outlet. In some embodiments, the outlet is proximate to reaction region or connected via a second set of conduits in fluid communication to the reaction region, the outlet also in fluid communication with fluid collection point. In some embodiments, the first reservoir, the second reservoir and / or the fluid collection point are removable vessels, such as tubes that can be filled, emptied, or otherwise manipulated, manually or automatically, prior to, contemporaneously with or after operation of the fluid circuit. In some embodiments, the removeable vessels are from about 5 to about 100 mL conical tubes with threaded caps that can be removed or attached via a receiving screw port at or proximate to an inlet positioned between the reservoir and the first set of conduits.

[0188] In some embodiments, the conduit connecting the first and second reservoir to the reaction region comprises at least a first input port and a second input port, the first and second input ports comprising a connection element through which the removable reservoir is attached to the fluid circuit. In some embodiments, the first and second input port is a screw port comprising thread cavity that receives a screw top from a removeable vessel, such as a conical tube.

[0189] In some embodiments, the system comprises at least a first output port; a flow path interconnecting the first input port, the second input port, and the first output port; at least a first switch valve downstream of the first input port and upstream of the first output port, and at least a second switch valve downstream of the second input port and upstream of the first output port. In some embodiments, the system comprises a mixing element in the reaction region, which isATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION downstream of the first and second switch valves and upstream of the first output port; wherein the first switch valve is switchable between at least a first operable state and a second operable state, wherein, in the first operable state, the first switch valve allows the first input port to be fluidly connected via the flow path of the fluid circuit to the reaction region; and wherein, in the second operable state, the first switch valve prevents the first input port from being fluidly connected to the reaction region; and wherein the second switch valve is switchable between at least a first operable state and a second operable state; wherein in the first operable state the second switch valve allows the second input port to be fluidly connected via the flow path to the reaction region; and wherein in the second operable state the second switch valve prevents the second input port from being fluidly connected to the reaction region. In some embodiments, the reaction region comprises a first mixing feature, such as a micro-impeller or cavity within which or over which tangential flow of fluid in the fluid circuit causes fluid mixing from the first set of conduits to mix. In some embodiments, the system comprises one or more controllers operably linked to the one or tplurality of pumps and configured to control the states of the first and second switch valves such that: when the first switch valve is in the first operable state, the controller controls the second switch valve to be in the second operable state; and when the first switch valve is in the second operable state, the controller controls the second switch valve to be in the first operable state. In embodiments, the one or more controllers comprise a dedicated controller for each of the first and second switch valves. In further embodiments, the dedicated switch controllers are programmable separately or as a group.

[0190] The disclosure also provides a system including: a third switch valve downstream of the first mixing feature and upstream of the first output port, wherein the third switch valve is switchable between at least a first operable state and a second operable state, wherein in the first operable state the third switch valve allows the reaction region to be fluidly connected via the flow path to the first output port, and wherein, in the second operable state, the third switch valve prevents the first mixing feature from being fluidly connected to the first output port. In some embodiments, the system comprises a mixing feature in the reaction region, and the fluid circuit further comprises at least a second set of conduits downstream of the reaction region leading to a waste output port. In some embodiments, there is a third switch valve downstream of the reaction region and upstream of the waste output port, wherein the third switch valve is switchable between at least a first operable state and a second operable state; wherein, in the first operable state, theATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION third switch valve allows the reaction region to be fluidly connected via the flow path to the waste output port, and wherein in the second operable state the third switch valve prevents the reaction region from being fluidly connected to the waste output port.

[0191] In some embodiments, a third input port is interconnected to the first output port by the flow path of the fluid circuit. In some embodiments, the third input port is upstream of the first reaction region. In still other embodiments, there is a third switch valve downstream of the third input port and upstream of the first output port, wherein the third switch valve is switchable between at least a first operable state and a second operable state, wherein in the first operable state the third switch valve allows the third input port to be fluidly connected via the flow path to a mixing feature in the reaction region, and wherein in the second operable state the third switch valve prevents the third input port from being fluidly connected to a mixing feature in the reaction region.

[0192] In embodiments, the system further comprises one or more controllers configured to control the states of the first and third switch valves such that: when the first switch valve is in the first operable state, the controller controls the third switch valve to be in the first operable state; and when the first switch valve is in the second operable state, the controller controls the third switch valve to be in the second operable state. In embodiments, the first and third input ports are for the introduction of materials (such as an aqueous solution), and the second input port is for the introduction of a clearing buffer. In embodiments, the output port is for the exit of materials having been mixed in the reaction region. In embodiments, at least one of the first and second switch valves comprises a compression / diaphragm valve. In still other embodiments, at least one of the first and second switch valves comprises a valve selected from a group consisting of: a socket valve; a rocker valve; a flipper valve; a plunger valve; a capillary valve; and a ball valve.

[0193] In some embodiments, at least one of the first and second switch valves is switchable between the first operable state and the second operable state in response to volumetric pressure. In embodiments, the first and second switch valves is switchable between the first operable state and the second operable state in response to pneumatic pressure . In embodiments, at least one of the first and second switch valves is switchable between the first operable state and the second operable state by a solenoid. In embodiments, there is a third switch valve downstream of the first input port and upstream of the output port; a fourth switch valve downstream of the second input port and upstream of the output port; and a second reaction region downstream of theATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION third and fourth switch valves and upstream of the output port, wherein the third switch valve is switchable between at least a first operable state and a second operable state, wherein in the first operable state the third switch valve allows the first input port to be fluidly connected via the flow path to the second reaction region (optionally comprising a second mixing feature), and wherein in the second operable state the third switch valve prevents the first input port from being fluidly connected to the second reaction region, and wherein the fourth switch valve is switchable between at least a first operable state and a second operable state, wherein in the first operable state the fourth switch valve allows the second input port to be fluidly connected via the flow path to the second reaction region, and wherein in the second operable state the fourth switch valve prevents the second input port from being fluidly connected to the second mixing feature.

[0194] In some embodiments, one or more controllers configured to control the states of the first, second, third, and fourth switch valves are provided, such that: when the first switch valve is in the first operable state, the controller controls the second and third switch valves to be in the second operable state, and controls the fourth switch valve to be in the first operable state; and when the first switch valve is in the second operable state, the controller controls the second and third switch valves to be in the first operable state, and controls the fourth switch valve to be in the second operable state.

[0195] In some embodiments of the invention, the first mixing feature comprises one or both of a Dean’s Vortex mixer and a herringbone mixer.

[0196] In some embodiments, the disclosure relates to a system that comprises at least one processor, a program storage, such as memory, for storing program code executable on the processor, and one or more input / output devices and / or interfaces, such as data communication and / or peripheral devices and / or interfaces. In some embodiments, the user device and computer system or systems are communicably connected by a data communication network, such as a Local Area Network (LAN), the Internet, or the like, which may also be connected to a number of other client and / or server computer systems. The user device and client and / or server computer systems may further include appropriate operating system software. In some embodiments, the system comprises a processor comprising a computer program product for calculating the alignment of known telomeric repeat sequences to the data from a sample registered with the computer program product. In some embodiments, the system comprises a device that interacts with one or more communication channels or mediums or links, such that alignment processes for telomereATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION sequences of a sample are compared to control sequences stored on a memory and shared with a network in operable communication with the device.

[0197] In some embodiments, components and / or units of the devices described herein may be able to interact through one or more communication channels or mediums or links, for example, a shared access medium, a global communication network, the Internet, the World Wide Web, a wired network, a wireless network, a combination of one or more wired networks and / or one or more wireless networks, one or more communication networks, an a-synchronic or asynchronous wireless network, a synchronic wireless network, a managed wireless network, a non-managed wireless network, a burstable wireless network, a non-burstable wireless network, a scheduled wireless network, a non-scheduled wireless network, or the like.

[0198] Discussions herein utilizing terms such as, for example, “processing,”“computing,” “calculating,” “determining,” or the like, may refer to operation(s) and / or process(es) of a computer, a computing platform, a computing system, or other electronic computing device, that manipulate and / or transform data represented as physical (e.g., electronic) quantities within the computer's registers and / or memories into other data similarly represented as physical quantities within the computer’s registers and / or memories or other information storage medium that may store instructions to perform operations and / or processes. In some embodiments, the system comprises a controller, a processor and a computer program product with computerexecutable instructions for (a) setting a fluid flow rate within the fluid circuit, optionally via one or more pumps in operable connection to the fluid circuit; and / or (b) calculating a fluid flow rate within the disclosed fluid circuit. In some embodiments, methods of the disclosure comprises calculating a fluid flow rate in the system; setting a fluid flow rate in the disclosed system and exposing the disclosed lipid mixture to the an aqueous solution comprising the DNA molecule disclosed herein to spontaneously form one or a population of lipid nanoparticles. In some embodiments, the step of exposing is performed in the reaction region of the fluid circuit. In some embodiments, a step of initiating fluid flow in a first and second reservoir comprising the lipid mixture and aqueous solution, respectively, is performed before the step of exposing.

[0199] Some embodiments may take the form of an entirely hardware embodiment, an entirely software embodiment, or an embodiment including both hardware and software elements. Some embodiments may be implemented in software, which includes but is not limited to firmware, resident software, microcode, or the like. In some embodiments, the software elementATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION comprises instructions for performing at least one step of a computer-implemented method of manufacturing a lipid nanoparticle. In some embodiments, the instruction comprise a step of setting a fluid flow rate within any one of the disclosed fluid circuits.

[0200] Furthermore, some embodiments may take the form of a computer program product accessible from a computer-usable or computer-readable medium providing program code for use by or in connection with a computer or any instruction execution system. For example, a computer-usable or computer-readable medium may be or may include any apparatus that can contain, store, communicate, propagate, or transport the program for use by or in connection with the instruction execution system, apparatus, or device.

[0201] In some embodiments, the medium may be or may include an electronic, magnetic, optical, electromagnetic, InfraRed (IR), or semiconductor system (or apparatus or device) or a propagation medium. Some demonstrative examples of a computer-readable medium may include a semiconductor or solid-state memory, magnetic tape, a removable computer diskette, a Random Access Memory (RAM), a Read-Only Memory (ROM), a rigid magnetic disk, an optical disk, or the like. Some demonstrative examples of optical disks include Compact Disk-Read-Only Memory (CD-ROM), Compact Disk-Read / Write (CD-R / W), DVD, or the like.

[0202] In some embodiments, a data processing system suitable for storing and / or executing program code may include at least one processor coupled directly or indirectly to memory elements, for example, through a system bus. The memory elements may include, for example, local memory employed during actual execution of the program code, bulk storage, and cache memories which may provide temporary storage of at least some program code in order to reduce the number of times code must be retrieved from bulk storage during execution.

[0203] In some embodiments, input / output or I / O devices (including but not limited to keyboards, displays, pointing devices, etc.) may be coupled to the system either directly or through intervening VO controllers. In some embodiments, network adapters may be coupled to the system to enable the data processing system to become coupled to other data processing systems or remote printers or storage devices, for example, through intervening private or public networks. In some embodiments, modems, cable modems and Ethernet cards are demonstrative examples of types of network adapters. Other suitable components may be used.

[0204] Some embodiments may be implemented by software, by hardware, or by any combination of software and / or hardware as may be suitable for specific applications or inATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION accordance with specific design requirements. Some embodiments may include units and / or subunits, which may be separate of each other or combined together, in whole or in part, and may be implemented using specific, multi-purpose or general processors or controllers. Some embodiments may include buffers, registers, stacks, storage units and / or memory units, for temporary or long-term storage of data or in order to facilitate the operation of particular operable states or hardware components operably linked to the disclosed fluid circuits.

[0205] Some embodiments may be implemented, for example, using a machine-readable medium or article which may store an instruction or a set of instructions that, if executed by a machine, cause the machine to perform a method steps and / or operations described herein. Such machine may include, for example, any suitable processing platform, computing platform, computing device, processing device, electronic device, electronic system, computing system, processing system, computer, processor, or the like, and may be implemented using any suitable combination of hardware and / or software. The machine-readable medium or article may include, for example, any suitable type of memory unit, memory device, memory article, memory medium, storage device, storage article, storage medium and / or storage unit; for example, memory, removable or non-removable media, erasable or non-erasable media, writeable or re-writeable media, digital or analog media, hard disk drive, floppy disk, Compact Disk Read Only Memory (CD-ROM), Compact Disk Recordable (CD-R), Compact Disk Re-Writeable (CD-RW), optical disk, magnetic media, various types of Digital Versatile Disks (DVDs), a tape, a cassette, or the like. The instructions may include any suitable type of code, for example, source code, compiled code, interpreted code, executable code, static code, dynamic code, or the like, and may be implemented using any suitable high-level, low-level, object-oriented, visual, compiled and / or interpreted programming language, e.g., C, C++, Java™, BASIC, Pascal, Fortran, Cobol, assembly language, machine code, or the like.

[0206] The system may comprise an electrical circuit implemented as a hardware circuit comprising custom very-large-scale integration (VLSI) circuits or gate arrays, off-the-shelf semiconductors such as logic chips, transistors, or other discrete components. A circuit may also be implemented in programmable hardware devices such as field programmable gate arrays, programmable array logic, programmable logic devices or the like.

[0207] In some embodiments, the system comprises an electrical circuit operably linked to a hardware component of the system, such as a pump in operable connection to the fluid circuit.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONIn some embodiments, the system comprises a processor and a computer program product in machine-readable medium for execution by various types of processors. An identified electrical circuit of executable code may, for instance, comprise one or more physical or logical blocks of computer instructions, which may, for instance, be organized as an object, procedure, or function. Nevertheless, the executables of an identified circuit need not be physically located together, but may comprise disparate instructions stored in different locations which, when joined logically together, comprise the circuit and achieve the stated purpose for the circuit. Indeed, a circuit of computer readable program code may be a single instruction, or many instructions, and may even be distributed over several different code segments, among different programs, and across several memory devices. Similarly, operational data may be identified and illustrated herein within circuits, and may be embodied in any suitable form and organized within any suitable type of data structure. The operational data may be collected as a single data set, or may be distributed over different locations including over different storage devices, and may exist, at least partially, merely as electronic signals on a system or network.

[0208] The computer readable medium (also referred to herein as machine-readable media or machine-readable content) may be a tangible computer readable storage medium storing the computer readable program code. The computer readable storage medium may be, for example, but not limited to, an electronic, magnetic, optical, electromagnetic, infrared, holographic, micromechanical, or semiconductor system, apparatus, or device, or any suitable combination of the foregoing. As alluded to above, examples of the computer readable storage medium may include but are not limited to a portable computer diskette, a hard disk, a random access memory (RAM), a read-only memory (ROM), an erasable programmable read-only memory (EPROM or Flash memory), a portable compact disc read-only memory (CD-ROM), a digital versatile disc (DVD), an optical storage device, a magnetic storage device, a holographic storage medium, a micromechanical storage device, or any suitable combination of the foregoing. In the context of this document, a computer readable storage medium may be any tangible medium that can contain, and / or store computer readable program code for use by and / or in connection with an instruction execution system, apparatus, or device.

[0209] The computer readable medium may also be a computer readable signal medium. A computer readable signal medium may include a propagated data signal with computer readable program code embodied therein, for example, in baseband or as part of a carrier wave. Such aATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION propagated signal may take any of a variety of forms, including, but not limited to, electrical, electro-magnetic, magnetic, optical, or any suitable combination thereof. A computer readable signal medium may be any computer readable medium that is not a computer readable storage medium and that can communicate, propagate, or transport computer readable program code for use by or in connection with an instruction execution system, apparatus, or device. As also alluded to above, computer readable program code embodied on a computer readable signal medium may be transmitted using any appropriate medium, including but not limited to wireless, wireline, optical fiber cable, Radio Frequency (RF), or the like, or any suitable combination of the foregoing. In one embodiment, the computer readable medium may comprise a combination of one or more computer readable storage mediums and one or more computer readable signal mediums. For example, computer readable program code may be both propagated as an electro-magnetic signal through a fiber optic cable for execution by a processor and stored on RAM storage device for execution by the processor.

[0210] Computer readable program code for carrying out operations for aspects of the present disclosure may be written in any combination of one or more programming languages, including an object-oriented programming language such as Java, Smalltalk, C++ or the like and conventional procedural programming languages, such as the “C” programming language or similar programming languages. The computer readable program code may execute entirely on a user's computer, partly on the user’ s computer, as a stand-alone computer-readable package, partly on the user’s computer and partly on a remote computer or entirely on the remote computer or server. In the latter scenario, the remote computer may be connected to the user’s computer through any type of network, including a local area network (LAN) or a wide area network (WAN), or the connection may be made to an external computer (for example, through the Internet using an Internet Service Provider).

[0211] The program code may also be stored in a computer readable medium that can direct a computer, other programmable data processing apparatus, or other devices to function in a particular manner, such that the instructions stored in the computer readable medium produce an article of manufacture including instructions which implement the function / act specified in the schematic flowchart diagrams and / or schematic block diagrams block or blocks.

[0212] Functions, operations, components and / or features described herein with reference to one or more embodiments, may be combined with, or may be utilized in combination with, oneATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION or more other functions, operations, components and / or features described herein with reference to one or more other embodiments, or vice versa.

[0213] In some embodiments, the system comprises a controller operably linked to the pump and / or pumps and one or more wireless communication components. In embodiments, the one or more wireless communication components comprise one or more radiofrequency identification components.Methods

[0214] LNP -formulated DNA is likely impacted by the association between lipid amine groups to nucleic acid backbone phosphates (N / P ratio). The N / P ratio is calculated with the following formula:N / P Ratio = (# Mol Anionic Lipid * # Tertiary Amine) / (# Mol plasmid DNA * # phosphate)

[0215] 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. 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. To see whether this relationship could impact immunogenicity via intramuscular delivery, mice were immunized with about 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. Interestingly, there was a significant attenuation in the frequency of total GC and HA-specific GC B with a formulation at 10: 1, but not 20: 1. These data suggest that the relationship between lipid and DNA could impact adaptiveATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION immunity with antigen-specific B cell responses more sensitive than Tfh priming; higher lipid to DNA ratios lead to improved immune responses.

[0216] The disclosure relates to a method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, the method comprising:(a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 20 to about 1, respectively. In some embodiments, the lipid nanoparticles are free of RNA encoding a therapeutic protein. In some embodiments, the lipid nanoparticles are free of RNA and the method is free of TLR9 stimulation in the subject.

[0217] In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 39 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 39 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 38 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 37 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 36 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administeringATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 35 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 34 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 33 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 32 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 31 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 30 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 29 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 28 to about 1. In some embodiments, the method of reducing an antigen-specific immuneATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 27 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 26 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 25 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 24 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 23 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 22 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 19 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower thanATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION about 18 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 17 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 16 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 15 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 14 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 13 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 12 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio lower than about 11 to about 1. In some embodiments, the method of reducing an antigen-specific immune response against a gene therapy vector in a subject in need thereof, comprises: (a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding aATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION therapeutic protein, wherein the lipid nanoparticles have a lipid to DNA weight ratio equal to or lower than about 10 to about 1.

[0218] In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 5 mb per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule, wherein the lipid mixture comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 6 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 7 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 8 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 9 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in aATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION fluid circuit under a flow rate of from about 10 mb per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 11 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 12 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 13 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 14 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule. In some embodiments, the method of reducing an antigen-specific immune response further comprises a step of manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 15 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule.

[0219] In some embodiments, the lipid mixture used in the method further comprises:(i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; andATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION(iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about1.5, respectively.

[0220] In some embodiments, the N / P ratio in a method herein is about 0.5, about 1, about1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about19.5, or about 20. In some embodiments, the N / P ratio in the method is 0.5, 1, 1.5, 2, 2.5, 2.6, 3,3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.0, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from about 0.5 to about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 2 to about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 2.6 to about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 4 to about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 5.3 to about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION about 10, about 10.5, about 1 1, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 7 to about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 9 to about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 10.5 to about 11, about11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from about 15 to about 15.5, about 16, about16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the method is from 0.5 to 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3,5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5,17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 2 to2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 2.6 to 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 4 to 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5,10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 5.3 to 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 7 to 7.5, 8, 8.5, 9, 9.5, 10, 10.5,11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 9 to 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 10.5 to 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the method is from 15 to 15.5, 16,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. Tn some embodiments, the N / P ratio in the method is about 2.6, about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the method is from about 2.6 to about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the method is from about 5.3 to about 10.5, or about 15. In some embodiments, the N / P ratio in the method is from about 10.5 to about 15. In some embodiments, the N / P ratio in the method is about 2.6 to about 5.3. In some embodiments, the N / P ratio in the method is about 10.5 to about 15. In some embodiments, the N / P ratio in the method is equal to or less than about 15. In some embodiments, the N / P ratio in the method is equal to or less than about 10.5. In some embodiments, the N / P ratio in the method is equal to or less than about 5.3. In some embodiments, the N / P ratio in the method is equal to or less than about 2.6. In some embodiments, the N / P ratio in the method is equal to or greater than about 2.6. In some embodiments, the N / P ratio in the method is equal to or greater than about 5.3. In some embodiments, the N / P ratio in the method is equal to or greater than about10.5. In some embodiments, the N / P ratio in the method is equal to or greater than about 15. In some embodiments, the NZP ratio in the method is 2.6, 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the method is from 2.6 to 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the method is from 5.3 to 10.5, or 15. In some embodiments, the N / P ratio in the method is from 10.5 to 15. In some embodiments, the N / P ratio in the method is 2.6 to 5.3. In some embodiments, the N / P ratio in the method is 10.5 to 15. In some embodiments, the N / P ratio in the method is equal to or less than 15. In some embodiments, the N / P ratio in the method is equal to or less than 10.5. In some embodiments, the N / P ratio in the method is equal to or less than 5.3. In some embodiments, the N / P ratio in the method is equal to or less than 2.6. In some embodiments, the N / P ratio in the method is equal to or greater than 2.6. In some embodiments, the N / P ratio in the method is equal to or greater than 5.3. In some embodiments, the N / P ratio in the method is equal to or greater than 10.5. In some embodiments, the N / P ratio in the method is equal to or greater than 15.

[0221] In some embodiments, the disclosure relates to a method of manufacturing or loading an LNP with an amount of nucleic acid molecule based upon an N / P ratio. In some embodiments, the method comprises exposing the nucleic acid molecule to a lipid mixture comprising any lipid species or combination herein. In some embodiments, the lipid mixture comprises (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof; and (iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivativeATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the N / P ratio is any one of the above N / P ratios or an N / P ratio within a range of N / P ratios about (endpoints inclusive). In some embodiments, the LNP is for reducing immune response and the N / P ratio is a ratio above that is less than or equal to about 10.5, less than or equal to about 5.3, or less than or equal to about 2.6 or a ratio in a range of ratios above (endpoints inclusive) where the maximum N / P ratio is less than or equal to about 10.5, less than or equal to about 5.3, or less than or equal to about 2.6. In some embodiments, the LNP is for providing gene expression while reducing immune response and the N / P ratio is a ratio above that is less than or equal to about 10.5, less than or equal to about 5.3, or less than or equal to about 2.6 or a ratio in a range of ratios above (endpoints inclusive) where the maximum N / P ratio is less than or equal to about 10.5, less than or equal to about 5.3, or less than or equal to about 2.6. In some embodiments, the LNP is for improving B-cell response and the N / P ratio is a ratio above that is greater than or equal to about 10.5 or is a ratio in a range of ratios above (endpoints inclusive) where the minimum N / P ratio is greater than or equal to about 10.5.

[0222] The disclosure also relates to a method of selectively loading lipid nanoparticles with a certain mass amount of DNA comprising: adjusting flow rate in a fluid circuit to arrive at a substantially homogeneous population of lipid nanoparticles with a desired N / P ratio; wherein, if the desired N / P ratio is from about 5 to about 20, setting the flow rate in the fluid circuit to at least about 5 mL per minute. In some embodiments, if the N / P ration is equal to or above about 10, the method further comprises filtering the population of lipid nanoparticles to remove lipid nano particles that are not encapsulating a DNA molecule. In some embodiments, the disclosure relates to a method of selectively loading lipid nanoparticles with a mass amount of DNA comprising: (a) choosing a desired N / P ratio of the lipid nanoparticle; and (b) adjusting flow rate in a fluid circuit to arrive at a substantially homogeneous population of lipid nanoparticles with the desired N / P ratio; wherein, if the desired N / P ratio is from about 1 to about 8, setting the flow rate of at least about 5 mL per minute; wherein the method further comprises: (c) exposing a composition comprising a lipid mixture and a DNA molecule in the fluid circuit; and (d) allowing a time period sufficient for the lipids to spontaneously form a population of lipid nanoparticles and encapsulate the DNA molecule; wherein the lipid mixture comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC) or a derivative thereof. In some embodiments, the lipid mixture further comprises: (i) SM-102 or a derivative thereof; (ii) a cholesterol molecule or a derivative thereof;ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION and (iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively. In some embodiments, the method comprises lipid nanoparticles with a molar ratio of components of any of the disclosed ratios herein.

[0223] In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 80% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 80% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 81% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 82% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 83% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 84% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 85% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 86% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 87% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 88% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 89% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 90% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 91% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticlesATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION comprising no less than about 92% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 93% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 94% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 95% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 96% of the lipid nanoparticles with the same or substantially similar total mass amount.

[0224] In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 97% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 98% of the lipid nanoparticles with the same or substantially similar total mass amount. In some embodiments, the method results in a population of lipid nanoparticles comprising no less than about 99% of the lipid nanoparticles with the same or substantially similar total mass amount.

[0225] In some embodiments, the certain total mass amount of DNA is from about 0.002 micrograms to about 4 micrograms. In some embodiments, 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.

[0226] Although the disclosure has been described with reference to exemplary embodiments, it is not limited thereto. Those skilled in the art will appreciate that numerous changes and modifications may be made to the preferred embodiments of the disclosure and that such changes and modifications may be made without departing from the true spirit of the disclosure. It is therefore intended that the appended claims be construed to cover all such equivalent variations as fall within the true spirit and scope of the disclosure. All referenced journal articles, patents, and other publications are incorporated by reference herein in their entireties.

[0227] The disclosure also relates to a method of manufacturing a population of LNPs by exposing a lipid mixture disclosed herein to an aqueous solution of a DNA molecule intended to be encapsulated by the lipid mixture. In some embodiments, the lipid mixture comprises from about 1% to about 10% ethanol and the lipid are suspended in an aqueous solution. In someATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has an fluid flow rate from about 5 mL per minute to about 20 mb per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has an fluid flow rate from about 4 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 6 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 7 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 8 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 9 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 10 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 11 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended forATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 12 mb per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 13 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has an a fluid flow rate from about 14 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has an a fluid flow rate from about 15 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate from about 16 mL per minute to about 20 mL per minute across a reaction region of the fluid circuit.

[0228] In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 4 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 5 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 6 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or pluralityATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 7 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 8 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 9 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules.

[0229] In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 10 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 11 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 12 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 13 mL per minute across a region of the fluid circuit in which the lipid mixture isATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 14 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of at least about 15 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules.

[0230] In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 200 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 175 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 150 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 125 mL per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 100 mL per minuteATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules. In some embodiments, the lipid mixture is exposed to the aqueous solution comprising a DNA molecule intended for encapsulation in a fluid circuit, the fluid circuit a component in any of the disclosed systems, whereby the fluid circuit has a fluid flow rate of from about 5 to about 75 mb per minute across a region of the fluid circuit in which the lipid mixture is exposed to the DNA molecule or plurality of DNA molecules.

[0231] The disclosure relates to the selectively reducing the magnitude of the immune response in a subject to a gene therapy vector by administering to the subject a gene therapy vector that is an LNP comprising a DNA molecule encoding a therapeutic agent; wherein the magnitude of the immune response is normalized against the magnitude of the immune response against the same gene therapy vector encapsulated in a LNP without the lipid to DNA mass ratios disclosed herein. In some embodiments, the magnitude of the immune response is normalized against the magnitude of the immune response against an RNA gene therapy vector encapsulated in an LNP.

[0232] The disclosure also relates to a method of modulating the amount of DNA loaded into or encapsulated into an LNP comprising, in a fluid circuit within a system disclosed herein, adjusting the fluid flow rate of a lipid mixture across a reaction region of the fluid circuit when the lipid mixture is exposed to the amount of DNA. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 40 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 45 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 50 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhancedATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 55 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 60 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 65 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 70 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 75 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, if a desired population of LNPs are administered to induce an enhanced immune response against an antigen in a subject in need thereof relative to the immune response associated with an RNA molecule encoding the antigen, a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 80 to about 1 is manufactured with a flow rate disclosed herein. In some embodiments, the LNP is manufactured by self-assembly of the LNP in the presence of the DNA in a fluid circuit with a flow rate of at least about 5 mL per minute across a reaction region of the fluid circuit.

[0233] The disclosure relates to a method of reducing the immune response against an LNP in a subject in need thereof comprising administering to the subject an LNP disclosed herein loadedATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION with an amount of DNA molecule disclosed herein, wherein the reduction of the immune response is relative to the immune response elicited by an LNP comprising an RNA.

[0234] The disclosure relates to a method of reducing the immune response against an LNP in a subject in need thereof comprising inducing the STING pathway and / or not inducing the TLR9 pathway in the subject. In some embodiments, the method comprises administering an LNP herein to a subject in need thereof. In some embodiments, the LNP comprises one or nucleic acid molecules. In some embodiments, the LNP is made by a method herein. In some embodiments, the N / P ratio in the LNP is about 0.5, about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is 0.5, 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.0, 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from about 0.5 to about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2 to about 2.5, about2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 4 to about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, aboutATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 5.3 to about 5.5, about 6, about 6.5, about 7, about 7.5, about8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 7 to about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 9 to about9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 15 to about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from 0.5 to 1, 1.5, 2, 2.5, 2.6, 3,3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5,15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2 to 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2.6 to 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5,9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 4 to 4.5, 5, 5.3, 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 5.3 to 5.5, 6, 6.5, 7,7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18,18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 7 to 7.5, 8, 8.5, 9,9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 9 to 9.5, 10, 10.5, 11, 11.5, 12, 12.5,ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 10.5 to 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 15 to 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is about 2.6, about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 5.3 to about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is about 2.6 to about 5.3. In some embodiments, the NZP ratio in the LNP is about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 15. In some embodiments, the N / P ratio in the LNP is 2.6, 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 2.6 to 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 5.3 to 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 10.5 to 15. In some embodiments, the N / P ratio in the LNP is 2.6 to 5.3. In some embodiments, the N / P ratio in the LNP is 10.5 to 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than 15.

[0235] In some embodiments, the method comprises administering the LNP comprising 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).ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0236] 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 non-contiguous alkyl or heteroalkyl chains covalently bound to a COO, wherein the first alkyl chain is from about 3 to about 5 contiguously bound carbon atoms in length and the second alkyl chain is from about 2 to about 14 carbon atoms in length. In some embodiments, n is 1, 2 or 3, and Ri is a C12 to C28 monoglyceride, alkenyl, alkyl, aryl, or aralkyl.

[0237] 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:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0238] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION

[0239] In some embodiments, the SM-102 derivative is a compound having Formula II, wherein Ri is chosen from one of the following:

[0240] 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.

[0241] In some embodiments, the disclosure relates to a method of stimulating the immune response in a subject in need thereof. In some embodiments, the method comprises administeringATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION an LNP comprising a nucleic acid molecule encoding an antigen or antigenic determinant thereof, wherein the N / P ration is from about 0.5 to about 20 and wherein the method is free of inducing TLR9 inflammatory response. In some embodiments, the method is free of inducing TLR9 and other pathways. In some embodiments, the LNP is any herein. In some embodiments, the LNP is made by a method herein. In some embodiments, the N / P ratio in the LNP is about 0.5, about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is 0.5, 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.0, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from about 0.5 to about 1, about 1.5, about 2, about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2 to about 2.5, about 2.6, about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 3, about 3.5, about 4, about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 4 to about 4.5, about 5, about 5.3, about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from aboutATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION5.3 to about 5.5, about 6, about 6.5, about 7, about 7.5, about 8, about 8.5, about 9, about 9.5, about10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 7 to about 7.5, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 9 to about 9.5, about 10, about 10.5, about11, about 11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 11, about11.5, about 12, about 12.5, about 13, about 13.5, about 14, about 14.5, about 15, about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from about 15 to about 15.5, about 16, about 16.5, about 17, about 17.5, about 18, about 18.5, about 19, about 19.5, or about 20. In some embodiments, the N / P ratio in the LNP is from 0.5 to 1, 1.5, 2, 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17,17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2 to 2.5, 2.6, 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14,14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 2.6 to 3, 3.5, 4, 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12,12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 4 to 4.5, 5, 5.3, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5,12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 5.3 to 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 7 to 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5,13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 9 to 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16,16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20. In some embodiments, the N / P ratio in the LNP is from 10.5 to 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONIn some embodiments, the N / P ratio in the LNP is from 15 to 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19,19.5, or 20. In some embodiments, the N / P ratio in the LNP is about 2.6, about 5.3, about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 2.6 to about 5.3, about10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 5.3 to about 10.5, or about 15. In some embodiments, the N / P ratio in the LNP is from about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is about 2.6 to about 5.3. In some embodiments, the N / P ratio in the LNP is about 10.5 to about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 15. In some embodiments, the N / P ratio in the LNP is equal to or less than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than about 15. In some embodiments, the N / P ratio in the LNP is 2.6, 5.3,10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 2.6 to 5.3, 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 5.3 to 10.5, or 15. In some embodiments, the N / P ratio in the LNP is from 10.5 to 15. In some embodiments, the N / P ratio in the LNP is 2.6 to 5.3. In some embodiments, the N / P ratio in the LNP is 10.5 to 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 15. In some embodiments, the N / P ratio in the LNP is equal to or less than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or less than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or less than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 2.6. In some embodiments, the N / P ratio in the LNP is equal to or greater than 5.3. In some embodiments, the N / P ratio in the LNP is equal to or greater than 10.5. In some embodiments, the N / P ratio in the LNP is equal to or greater than 15.EXAMPLESExample 1: Lipid Nanoparticle ManufacturingMethods

[0242] 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. Plasmid DNA- LNPs were formulated using microfluidic mixing of organic phase containing lipids and aqueousATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION phase containing plasmid DNA. Lipids were dissolved in ethanol at a molar ratio of 50:38.5: 10: 1 .5 (SM102 : Cholesterol : Distearoylphosphatidylcholine (DSPC) : DMG-Polyethelene glycol (DMG-PEG)). Lipids utilized include SM102 (Echelon Bioscience, Cat#N-1102), cholesterol (Avanti polar lipids, Cat#700000), DMG-PEG 2000 (Avanti polar lipids, Cat#880151), and 18:0 PC (DSPC; Avanti polar lipids, Cat#850365). Plasmid DNA was diluted in citrate buffer (citrate buffer 50 mM, pH-4) and mixed with lipid containing ethanol at a volumetric ratio of 1 :3 (ethanol: citrate buffer) using microfluidic mixing device (NanoAssembler Ignite, Precision Nanosystems). Formulations were synthesized at varying N / P ratios and using different flow rates (mL per minute). The LNPs were then dialysed against lx PBS buffer. The LNP formulations were characterized for their size using Zetasizer Pro ZS (Malvern Panalytical).

[0243] NanoFCMdata acquisition and analysis - DNA-LNP nanoparticles were concentrated to a particle concentration of approximately 108— 109particles / mL. LNPs were stained with SYTO9 (Catalog No. S34854) at a final concentration of 0.05 mM and incubated for 30 minutes at room temperature (25 °C). For instrument calibration, NanoFCM™ Silica Nanospheres Cocktail #! (CatalogNo. S16M-Exo; diameter: 68-155 nm) was used to generate a standard curve correlating particle size with side scatter intensity. Data acquisition for SYTO9-stained LNPs was performed on the FITC channel. Gating was applied to distinguish two populations: SYTO9- positive (loaded LNPs) and SYTO9-negative (empty LNPs). Size distribution analysis was performed using the derived calibration curve. Fluorescence intensity of SYTO9-positive LNPs and the fluorescence intensity of single free cargo molecules (free DNA) was determined by the NanoFCM software. Copy number distribution across the LNP population was calculated by dividing the fluorescence intensity of loaded LNPs by the fluorescence intensity of a single free cargo molecule.Results

[0244] To assess the impact of the N / P ratio and flow rate during formulation of DNA- LNPs, formulation was performed at varying N / P ratios ranging from 6 to 18 and at flow rates ranging from 3 to 20 mL per minute and particle size was measured. It was observed that at lower N / P ratios, flow rate impacts particle size, where higher flow rates tend to reduce particle size relative to low flow rates (FIGS. 1A-C). These observations persist using an N / P ratio of 6, 10.5, and 12. However, at a high N / P ratio of 15, the impact of flow rate on particle size is minimized, which minimal variability of particle size at all tested flow rates (Fig. ID). Taken together, theseATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION data demonstrate that the flow rate during particle formation can dramatically impact particle size at lower N / P ratios.

[0245] We next sought to determine the efficiency of plasmid DNA loading into the LNP using nano flow cytometry (NanoFCM). Previous studies of mRNA-LNPs have demonstrated that approximately 23% of LNPs are loaded with mRNA when formulated, and the average number of mRNA molecules per LNP is between 2 and 3. See Li S, Hu Y, Li A, Lin J, Hsieh K, Schneiderman Z, et al. "''Payload distribution and capacity of mRNA lipid nanoparticles I" Nature Communications. 2022; 13(1): 5561. Proportionally, DNA-LNPs made at N / P 6 have at most 20% of particles loaded with DNA as determined with NanoFCM (FIG. IE). Using SYBR Green, we determined that the majority of loaded LNPs had two copies of DNA (FIG. IF). DNA-LNPs made at N / P 10.5 had a larger fraction (>30%) of LNPs loaded with DNA at a flow rate of 3 and 6 mL per minute with a similar copy number profile (FIGS. 1G and 1H). DNA-LNPs made at N / P 15 and a flow rate of 6 ml per minute had greater than 40% of particles loaded with DNA, the majority containing 2 copies (FIGS. II and 1 J). These data demonstrate that CA09 HA DNA-LNPs at N / P 15 exhibit improved DNA loading, and that DNA-LNP flow rates and N / P ratios can be modulated to control for amount of DNA loading and copy number profile.Relationship between lipid components and DNA impacts immunogenicity

[0246] 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. 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. 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. To examine how this relationship impacts immunogenicity, mice were immunized with HA DNA-LNPs at different N / P ratios orATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION 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 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. In the T cell compartment, we observed a similar activated T follicular helper (Tfh) cell response with formulations at all three N / P ratios. 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 (or cytokine-expressing CD8+ or CD4+ T cells. 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.DNA-LNPs induce cGAS-STING pathway-dependent inflammation

[0247] 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. 8F-8G). 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.

[0248] 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 plasmidATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONDNA alone did not, suggesting that LNP -mediated delivery of plasmid DNA is driving innate immune inflammation (FIG. 8H-8I). However, in the presence of inhibitors to the double stranded DNA (dsDNA) sensing cGAS-STING-TBKl 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. 8J) is a driver of inflammatory responses to DNA-LNPs, while TLR9 sensing of CpG motifs is dispensable.

[0249] We sought to alter aspects of the platform to understand its contribution to immune responses. An increased NZP ratio has been associated with increased in vitro gene expression for RNA. Our weight ratio formulation of 40: 1 (an N / P ratio of 10.5: 1) improved B cell responses within the GC, implicating the lipid to DNA ratio as a modulator of immunogenicity in vivo. Based on these data, continued study of varied lipid to nucleic acid ratios for LNP-formulated genetic vaccines is important to further tailor immunogenicity of unique platforms for specific applications. Additionally, further understanding of available phosphate groups on plasmid DNA available for lipid binding remains important.

[0250] Example 2: TLR9 Activation

[0251] To assess whether DNA-LNPs induce TLR9 activation, mouse TLR9 reporter cells were incubated in the presence of DNA-LNPs, stimulatory mouse-preferred CpG ODN 1826, or inhibitory CpG ODN 2088 for 20 hours. Following incubation, TLR9 activation was measured using secreted embryonic alkaline phosphatase (SEAP). We observed no TLR9 activation at all concentrations of DNA-LNP measured in contrast to robust TLR9 activation with stimulatory CpG ODN 1826. See FIG. 9. These data suggest that DNA-LNPs do not induce TLR9 activation.

[0252] This experiment was conducted as follows. HEK-Blue mTLR9 cells (Invivogen) were cultured for three passages before assay start according to manufacturer’s instructions. Cells were placed under selection using DMEM supplemented with 4.5 g / 1 glucose, 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, 100 pg / mL streptomycin, 100 g / mL Normocin (Invivogen), 2mM L-glutamine, 30 pg / mL Blasticidin, and 100 pg / mL Zeocin. Cells were maintained at 37°C, 5% CO2. On the day of the assay, cells were resuspended in HEK-Blue Detection Medium (Invivogen) before culturing with DNA-LNP, CpG ODN 1826 (Invivogen), or CpG ODN 2088 (Invivogen). Assay was performed according to manufacturer’s instructions. CellsATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION were incubated for 20 hours at 37°C, 5% CO2 before reading at 620nm on BioTek Synergy Plate Reader.

Claims

ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATIONCLAIMS1. A method of making a lipid nanoparticle comprising exposing a composition comprising a lipid mixture and a deoxyribonucleic acid (DNA) molecule in a fluid circuit under a fluid flow rate of at least about 5 mL per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule, wherein the lipid mixture comprises 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC) or a derivative thereof.

2. The method of claim 1, wherein the composition further comprises:(i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; and(iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

3. The method of claims 1 or 2, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding one or a plurality of therapeutic agents.

4. The method of any of claims 1 through 3, wherein the lipid nanoparticle further comprises a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms.

5. The method of any of claims 1 through 4, 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.

6. The method of any of claims 2 through 5, wherein the polyethylene glycol is DMG-PEG- 2000 or a derivative thereof7. The method of any of claims 1 through 6, wherein the DNA molecule is a cDNA molecule, a linear DNA molecule, a circular plasmid DNA molecule, a mini-circle DNA molecule, a rollingATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION circle amplified DNA product, an artificial chromosome, a replicating DNA or any combination thereof.

8. The method of any of claims 1 through 7, wherein the lipid nanoparticle comprises a spheroid shape or semi-spheroid shape, and wherein the lipid nanoparticle comprises from about 50 to about 180 nanometers at its longest width dimension after spontaneous formation.

9. The method of any of claims 1 through 8 further comprising repeating the step of exposing, such that a population of lipid nanoparticles is manufactured and the lipid nanoparticles encapsulate one or a plurality of DNA molecules; and wherein the population of lipid nanoparticles are homogenous in shape and comprise from about 1 to about 10,000 nucleic acid molecules.

10. The method of any of claims 1 through 9, wherein the lipid nanoparticle or a population of lipid nanoparticles has a zeta potential of from about -2 millivolts to about -22 millivolts.

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

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

13. The method of any of claims 1 through 12, wherein the composition and the lipid nanoparticle are free of RNA.

14. The method of claim 2, wherein the method further comprises a step of mixing the lipid, the cholesterol, the polyethylene glycol and the SM-102 in an aqueous solution prior to exposing the lipid mixture to the DNA molecule.

15. The method of any of claims 1 through 14, wherein the step of exposing is performed within a fluid circuit with the flow rate in a lateral flow of solution under room temperature.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION16. The method of any of claims 1 through 15, wherein the step of exposing is performed on a system comprising a first reservoir, a second reservoir; and a pump, each of the first and second reservoirs connected to a fluid circuit and the pump configured to create fluid flow in the fluid circuit; wherein the fluid circuit comprises a first set of conduits connecting the first reservoir and the second reservoir to a reaction region.

17. The method of claim 16, wherein the system further comprises a processor, controller and computer program product; wherein the method is a computer-implemented method and the computer-program product comprises computer-executable instructions for:(a) initiating fluid flow from the first and second reservoirs; and(b) setting a fluid flow rate in the fluid circuit.

18. The method of claim 17, wherein the computer program product comprises computerexecutable instructions for (c) calculating the fluid flow rate in the fluid circuit.

19. The method of any of claims 1 through 18, wherein the fluid flow rate is from about 5 mL per minute to about 20 mL per minute.

20. A computer-implemented method of manufacturing a population of lipid nanoparticles in a system comprising a first reservoir, a second reservoir; and a pump, each of the first and second reservoirs connected to a fluid circuit and the pump configured to create fluid flow in the fluid circuit, the method comprising:(a) initiating fluid flow from the first reservoir into the fluid circuit;(b) initiating fluid flow from the second reservoir into the fluid circuit; and(c) exposing a composition comprising a lipid mixture and a deoxyribonucleic acid (DNA) molecule in a fluid circuit under a flow rate of from about 5 mL per minute to about 20 mL per minute; and allowing a time period sufficient for the lipids to spontaneously form a population of lipid nanoparticles and encapsulate the DNA molecule; wherein the lipid mixture comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof;ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION wherein the first reservoir comprises the lipid mixture and the second reservoir comprises an aqueous solution comprising the DNA molecule.

21. The method of claim 20, wherein the lipid mixture further comprises:(i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; and(iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

22. The method of any of claims 20 or 21, wherein the lipid nanoparticle further comprises a plurality of nucleic acid molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms.

23. The method of any of claims 20 through 22, 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.

24. The method of any of claims 20 through 23, wherein the polyethylene glycol is DMG- PEG-2000 or a derivative thereof25. A method of modulating an immune response in a subject in need thereof comprising:(a) administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio equal to or greater than about 40 to about 1 if enhancing an immune response in the subject; or(b) administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio lower than about 40 to about 1 if reducing an immune response in the subject.

26. The method of claim 25, wherein the DNA comprises a nucleotide sequence encoding one or more therapeutic agents.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION27. A method of delivering a therapeutic agent to a subject by inducing the STING pathway independent of and / or free of stimulating TLR9 comprising administering to the subject a population of lipid nanoparticles comprising DNA with a lipid to DNA weight ratio from about 6: 1 to about 20: 1.

28. The method of claim 27, wherein the average N / P ratio of the population of lipid nanoparticles is from about 4 to about 10.

29. A method of reducing an antigen-specific immune response to a gene therapy vector in a subject in need thereof, the method comprising:(a) administering to the subject a population of lipid nanoparticles comprising DNA gene therapy vector encoding a therapeutic protein, wherein the lipid nanoparticle have a lipid to DNA weight ratio lower than about 20 to about 1.

30. The method of claim 29 wherein the lipid nanoparticles are free of RNA encoding a therapeutic protein.

31. The method of any of claims 29 or 30, wherein the lipid nanoparticles are free of RNA and the method is free of TLR9 stimulation in the subject.

32. The method of any of claims 29 through 31 further comprising manufacturing the population of lipid nanoparticles prior to the step of administering by exposing a composition comprising a lipid mixture and a DNA molecule in a fluid circuit under a flow rate of from about 5 mb per minute to about 20 mb per minute; and allowing a time period sufficient for the lipids to spontaneously encapsulate the DNA molecule, wherein the lipid mixture comprises 1,2-distearoyl- sn-glycero-3 -phosphocholine (DSPC) or a derivative thereof.

33. The method of claim 32, wherein the composition further comprises:(i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; and(iii) polyethylene glycol or a derivative thereof;ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

34. A method of selectively loading lipid nanoparticles with a mass amount of DNA comprising:(a) choosing a desired N / P ratio of the lipid nanoparticle; and(b) adjusting flow rate in a fluid circuit to arrive at a substantially homogeneous population of lipid nanoparticles with the desired N / P ratio; wherein, if the desired N / P ration is from about 1 to about 8, setting the flow rate of at least about 5 mL per minute; and(c) exposing a composition comprising a lipid mixture and a DNAjmolecule in the fluid circuit; and(d) allowing a time period sufficient for the lipids to spontaneously form a population of lipid nanoparticles and encapsulate the DNA molecule; wherein the lipid mixture comprises l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or a derivative thereof.

35. The method of claim 34, wherein the lipid mixture further comprises:(i) SM-102 or a derivative thereof;(ii) a cholesterol molecule or a derivative thereof; and(iii) polyethylene glycol or a derivative thereof; wherein (i), (ii), the DSPC or derivative thereof, and (iii) are present at a molar ratio of about 50: about 38.5: about 10; and about 1.5, respectively.

36. The method of any of claims 34 or 35, wherein no less than 80% of the lipid nanoparticles in the population of lipid nanoparticles further comprises a plurality of DNA molecules with a total mass amount of from about 0.002 micrograms to about 4 micrograms.

37. The method of any of claims 34 through 36, 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.ATTORNEY DOCKET: WIST-016-PCT PATENT APPLICATION38. The method of any of claims 34 through 37, wherein the polyethylene glycol is DMG-PEG-2000 or a derivative thereof.

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