Lipid nanoparticle compositions for plasmid DNA delivery to the liver and methods for their preparation - Patents.com

Abstract

Lipid nanoparticle formulations that have cell type-specific transfection activity and are capable of generating Th1 and / or Th2 responses in vivo, and their use for plasmid DNA or mRNA delivery, are disclosed.

Description

STATEMENT OF GOVERNMENT RIGHTS

[0001] This invention was made with Government support under Grants A1155313 and EB028239 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Background technology]

[0002] Development of delivery systems and methods remains the most important challenge in realizing the tremendous potential of nucleic acid delivery for gene therapy. RNA and DNA-based biology have extensive capabilities to modulate cellular activities for the treatment of genetic and acquired diseases (Mulligan, 1993). Among non-viral gene delivery vectors, the clinical success of LNPs has recently attracted widespread attention (Witzigmann et al., 2020; Cullis and Hope, 2017). This is reflected in the development of the US Food and Drug Administration (FDA)-approved short interfering RNA therapy for hereditary amyloidosis (ONPATTRO). (R) (Onpattro, (R) This is highlighted by the two mRNA COVID-19 vaccines that have been approved or authorized for emergency use by millions of healthy people during the pandemic (Adams et al., 2018; Akinc et al., 2019; Hou et al., 2021).

[0003] Most lipid-based nucleic acid delivery platforms that have undergone clinical investigation or are commercially available consist of four or five components: ionic lipids, cholesterol, PEGylated lipids, helper phospholipids (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), and selective organ-targeting lipids (Cheng et al., 2020; Zhang et al., 2021; Wang et al., 2015; Cheng and Lee, 2016). Recent studies have reported that the choice of lipid components, as well as the relative proportions of lipid components in the formulation, greatly affect in vivo transfection efficiency and tissue-specific delivery (Cheng et al., 2020; Wei et al., 2020; Oberli et al., 2017; Li et al., 2015; Lokugamage et al., 2021).

[0004] Despite the verified ability of these formulations to encapsulate mRNA or siRNA and mediate cellular uptake and endosomal escape, there is a lack of detailed analysis of the impact of helper lipid charge and relative ratio of LNP components on transfection efficiency for plasmid DNA (pDNA) delivery, which can provide long-term transgene expression compared to mRNA (Kulkarni et al., 2017; Handumrongkul et al., 2019; Buck et al., 2019; Scholz and Wagner, 2012). In addition, the large number of candidate formulations to screen LNP systems for effective in vivo delivery makes it difficult to rationally determine the optimal formulation for a specific tissue or disease target. Summary of the Invention [Means for solving the problem]

[0005] In some embodiments, the presently disclosed subject matter provides solid nanoparticles including a nucleic acid payload comprising a steroid, an ionizable cationic lipid, a helper lipid, a PEGylated lipid, and one or more nucleic acids, wherein the nanoparticles comprise a molar ratio of steroid to PEGylated lipid of between about 10 and about 900; a molar ratio of ionizable cationic lipid to helper lipid of between about 1 and about 200; a total percentage of ionizable lipid and helper lipid of between about 20% and about 80%; and a P to N ratio of between about 2 and about 14.

[0006] In certain embodiments, the steroid comprises a sterol, hi particular embodiments, the sterol comprises cholesterol.

[0007] In certain embodiments, the ionizable cationic lipid includes Dlin-MC3-DMA.

[0008] In certain embodiments, the helper lipid is selected from a cationic lipid, a zwitterionic lipid, and an anionic lipid.

[0009] In certain embodiments, the cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and dimethyldioctadecylammonium (DDAB).

[0010] In certain embodiments, the zwitterionic lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0011] In certain embodiments, the anionic lipid comprises a phospholipid, in certain embodiments, the phospholipid is selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0012] In certain embodiments, the PEGylated lipid includes dimyristoylglycerol (DMG)-polyethylene glycol (PEG) 2000 (DMG-PEG2000).

[0013] In certain embodiments, the one or more nucleic acids are selected from plasmid DNA (pDNA), mRNA, siRNA, and combinations thereof. In some embodiments, the siRNA comprises an anti-inflammatory siRNA.

[0014] In another aspect, the presently disclosed subject matter provides a method for delivering one or more nucleic acids to the liver of a subject, the method comprising administering to a subject in need of treatment a solid nanoparticle as disclosed herein.

[0015] In certain embodiments, the one or more nucleic acids are selected from plasmid DNA (pDNA), siRNA, and combinations thereof.In particular embodiments, the one or more nucleic acids include a combination of plasmid DNA (pDNA) and siRNA.In even more particular embodiments, the siRNA includes anti-inflammatory siRNA.

[0016] In certain embodiments, the anti-inflammatory siRNA targets a transcription factor selected from signal transduction and activator of transcription (STAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ). In certain embodiments, the method further comprises reducing inflammation-induced gene silencing. In certain embodiments, the duration of expression of pDNA administered together with anti-inflammatory siRNA is longer than the duration of expression of pDNA administered alone. In certain embodiments, the expression level of pDNA administered together with anti-inflammatory siRNA is substantially similar to the expression level of pDNA administered alone.

[0017] In certain embodiments, the methods include reducing the levels in the liver of one or more signal transduction and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), one or more infiltrating inflammatory monocytes, and one or more apoptotic cells. In particular embodiments, the one or more infiltrating inflammatory monocytes are CD45 + and CD11b + Selected from cells.

[0018] In certain embodiments, the method includes treating one or more diseases or disorders of the liver. In particular embodiments, the one or more diseases or disorders of the liver are selected from genetic liver diseases and inflammatory liver diseases. In even more particular embodiments, the one or more diseases or disorders of the liver are selected from hemophilia B, hemophilia A, ornithine transcarbamylase (OTC) deficiency, phenylketonuria, acute intermittent porphyria, methylmalonic acidemia, familial hypercholesterolemia, Fabry, MPS type VI, gangliosidosis GM1, Danon disease, GSDla von Gierke, Wilson disease, Crigler-Najjar, primary hyperoxaluria type 1, and combinations thereof.

[0019] In certain embodiments, the method for delivering one or more nucleic acids to the liver of a subject is selected from intravenous (iv) injection, oral, subcutaneous, and inhalation delivery.

[0020] In another aspect, the presently disclosed subject matter provides a method for preparing a solid nanoparticle of the present disclosure, the method comprising: (a) preparing an organic phase by dissolving a mixture of steroid, ionizable cationic lipid, helper lipid, and PEGylated lipid in a predetermined molar ratio in a polar, protic solvent; (b) preparing an aqueous phase by dissolving one or more nucleic acids in an aqueous buffer; and (c) combining the organic phase and the aqueous phase to form solid nanoparticles.

[0021] In certain embodiments, the polar, protic solvent is a C1-C4 alcohol. In certain embodiments, the aqueous buffer comprises a magnesium acetate buffer.

[0022] In certain embodiments, the method further comprises mixing the organic phase and the aqueous phase in a flash nanocomposite formation (FNC) device.

[0023] In certain embodiments, the method further comprises combining the organic phase and the aqueous phase in a ratio of about 3:1.

[0024] In certain embodiments, the method further comprises dialysing the solid nanoparticles against deionized water.

[0025] In some embodiments, the present invention provides a method for stimulating T helper type 1 (Th1) and / or T helper type 2 (Th2) responses in vivo, the method comprising administering the solid nanoparticles disclosed in the present invention.

[0026] In certain embodiments, the steroid comprises cholesterol; the ionizable cationic lipid comprises DLin-MC3-DMA; the PEGylated lipid comprises DMG-PEG2000; the nucleic acid comprises mRNA; and the helper lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DSPC, 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0027] In certain embodiments, the solid nanoparticles comprise the following: a combined molar percentage of DLin-MC3-DMA and helper lipid ranging from about 20% to about 80%; a weight ratio of cholesterol to DMG-PEG2000 ranging from about 10 to about 500; a weight ratio of DLin-MC3-DMA to helper lipid ranging from about 1 to about 200; and a molar ratio of charged groups on the ionizable lipid to phosphate groups on the mRNA (N / P ratio) ranging from about 4 to about 12.

[0028] In certain embodiments, the solid nanoparticles comprise: (a) about 30 mol% DOPE, about 30 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 4; (b) about 7 mol% DSPC, about 70 mol% DLin-MC3-DMA, about 20 mol% cholesterol, about 0.04 mol% DMG-PEG2000, and an N / P ratio of about 4; or (c) about 5 mol% 18PG, about 55 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 12.

[0029] In certain embodiments, the methods induce an immune response in only Th1, only Th2, or both Th1 and Th2.

[0030] In another aspect, the present invention provides a method of treating a disease, disorder, or condition in a subject, comprising administering to a subject in need of treatment a therapeutically effective amount of the solid nanoparticles disclosed herein.

[0031] In certain embodiments, the disease is selected from cancer or infectious disease. In particular embodiments, the cancer is selected from basal cell carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal carcinoma, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, malignant pleural mesothelioma, Merkel cell carcinoma, metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer, squamous cell carcinoma, and urothelial carcinoma.

[0032] In certain embodiments, the infection comprises a viral infection, hi particular embodiments, the viral infection is selected from a coronavirus infection, a Zika virus infection, influenza, a flavivirus infection, and a human immunodeficiency virus (HIV) infection.

[0033] In other embodiments, the method further comprises administering the solid nanoparticles together with one or more immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitors are selected from CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In particular embodiments, the one or more immune checkpoint inhibitors are selected from ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0034] In other aspects, the presently disclosed subject matter provides vaccines comprising the solid nanoparticles disclosed herein, hi certain aspects, the vaccine is a cancer vaccine or an anti-viral vaccine.

[0035] Certain aspects of the presently disclosed subject matter have been set forth in the foregoing sections, which are addressed in whole or in part by the presently disclosed subject matter, and other aspects will become apparent as the description proceeds in conjunction with the accompanying examples and figures, which are best described herein below. [Brief description of the drawings]

[0036] The patent or application contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0037] Having described the subject matter of the present disclosure in general terms above, reference is now and again made to the accompanying figures, which are not necessarily drawn to scale. [Figure 1]Schematic diagram of multi-step compositional screening of lipid nanoparticles (LNPs) for liver-targeted pDNA delivery. 1,080 LNP formulations with different helper lipid and component ratios were evaluated for in vitro transfection efficiency. Top performing formulations for each helper lipid series were then tested in clusters for cytotoxicity and in vivo local transfection efficiency via intrahepatic injection. Clusters that induced minimal cytotoxicity and high transfection were screened via iv (intravenous) injection, and LNP formulations within clusters that demonstrated optimal liver transfection were further evaluated individually. [Diagram 2] Figures 2A, 2B, 2C, 2D, 2E, 2F, and 2G show in vitro LNP-mediated pDNA delivery. (Figure 2A) Transfection efficiency of LNPs in HepG2 cells via high-throughput screening platform after 72 h incubation (n=2). The level of transgene expression of each formulation is displayed using luciferase as a reporter. (Figure 2B) The top 32 formulations from each helper lipid series were selected based on transfection efficiency in HepG2. (Figure 2C-Figure 2E) (Figure 2C) FACS was used to further evaluate the transfection efficiency of LNPs of DOTAP, (Figure 2D) DOPE, and (Figure 2E) 18PG series using GFP as a reporter gene at a pDNA dose of 1 μg mL-1; gene expression was analyzed 72 h after transfection (n=2). Cell metabolic activity was measured by alamarBlue assay (n=4). Formulations were regrouped into 4 clusters containing 8 formulations each based on transgene expression levels. Data are presented as mean ± SD. The percentage of each component in the formulation is indicated by a pie chart. See Tables 1-6 for the molar percentages used in the 32 formulations of each helper lipid. (Figure 2F) Histogram of Z-average diameter of the top 32 LNP formulations made from each helper lipid. (Figure 2G) Percentage of LNP formulations with sizes less than 200 nm and less than 400 nm for each helper lipid. [Diagram 3] Figures 3A, 3B, 3C, 3D, and 3E show LNP-mediated local intrahepatic pDNA delivery in cluster mode screening. (Figure 3A) Scheme for intrahepatic delivery. (Figure 3B) Total bioluminescence flux in BALB / c mice at 12 hours after a single intrahepatic injection of different LNP formulations containing 3 μg pDNA (50% Luc + 50% mCherry) per mouse (n=2). (Figures 3C-3D) Ex vivo (Figure 3C) imaging and (Figure 3D) quantitative flux of luminescence in liver at 12 hours after administration. (Figure 3E) FACS was used to quantify the percentage of specific cell types expressing mCherry for the top 12 clusters in liver. Data are presented as mean ± SD (n=2 mice per group). [Figure 4] Figures 4A, 4B, 4C, 4D, 4E, 4F, 4G, 4H, and 4I show the in vivo transfection efficiency of LNPs administered via iv injection in cluster mode screening. (Figures 4A-4B) Quantitative measurement and (Figure 4B) imaging of total body bioluminescence in BALB / c mice 12 hours after a single iv administration of different LNPs formulations containing 100 μg pDNA (50% Luc + 50% mCherry) per mouse (n=2). (Figures 4C-4D) Ex vivo imaging and quantitative luminescence measurement of BALB / c mouse liver 12 hours after administration. (Figure 4E) FACS was used to quantify the percentage of specific cell types among mCherry+ cells in the liver. (Figure 4F) FACS was used to quantify the percentage of mCherry+ cells within hepatocytes (FSChi SSChi cells in CD45-CD31-CD11b-CD326- cells). (Figure 4G-Figure 4H) Quantitative measurements of luminescence and relative luciferase expression in each organ. (Figure 4I) FACS was used to quantify the percentage of mCherry+ cells within major organs. Data above are presented as mean ± SD. [Diagram 5]Figures 5A, 5B, 5C, 5D, 5E, 5F, 5G, 5H, 5I, 5J, 5K, and 5L show liver-targeted transfection efficiency and in vivo gene editing by LNPs administered via intravenous injection. (Figure 5A-Figure 5B) Whole-body bioluminescence imaging and quantitative measurement of BALB / c mice 12 hours after a single iv injection of different LNP formulations containing 50 μg pDNA (50% Luc + 50% mCherry) per mouse (n=2). (Figure 5C-Figure 5D) Ex vivo imaging and quantitative luminescence measurement of BALB / c mice liver 12 hours after a single administration. (Figure 5E-Figure 5F) Quantitative measurement of luminescence and relative luciferase expression levels in each organ. (Figure 5G) Schematic illustrating delivery of Cre pDNA activates tdTom expression in tdTom transgenic mice via Cre-mediated gene deletion of the stop cassette. (Fig. 5H) Ex vivo quantitative measurement of tdTom fluorescence in the liver of Ai9 mice 3 days after a single dose of LNPs containing 25 μg pDNA(Cre) per mouse (iv, n=2). (Fig. 5I) TdTom expression visualized by confocal imaging of tissue sections. (Fig. 5J) Ex vivo imaging of tdTom fluorescence in various organs of Ai9 mice 3 days after a single dose administration. (Fig. 5K) Relative expression of tdTom in each organ. (Fig. 5L) FACS was used to quantify the percentage of tdTom+ cells in the liver. All data are provided as mean ± standard deviation. [Figure 6]Figures 6A, 6B, 6C, 6D, 6E, 6F, 6G, and 6H show the in vivo distribution, cellular uptake, and endosomal escape levels of top performing LNP formulations. (Figure 6A) In vivo distribution at 6, 12, and 24 hours after injection of LNPs (30 μg pDNA (85% Luc + 15% Cy5-labeled p1216) per mouse, iv administration, n=3). (Figure 6B) FACS was used to quantify the percentage of Cy5+ hepatocytes in the liver 6 and 12 hours after injection. (Figure 6C-Figure 6D) Ex vivo quantitative measurement and luminescence imaging of BALB / c mouse liver 24 hours after administration (25 μg Luc pDNA per mouse, intrahepatic injection, n=3). (Figure 6E-Figure 6G) In vitro transfection and cellular uptake of selected formulations into primary hepatocytes. FACS was used to quantify the percentage of (Figure 6E) Cy5+ cells and (F) GFP+ cells in primary hepatocytes (1 μg mL-1 pDNA (75% GFP + 25% Cy5-labeled p1216)) isolated from the liver. (Figure 6G) Representative FACS data of LNPs pre-incubated with mouse serum for 0.5 h at a LNP / serum volume ratio of 2:1 before administration. (Figure 6H) Quantitative Cellomics high content analysis of endosomal escape mediated by LNPs in vitro. The average number of Gal8 spots per cell (B16-Gal8-GFP) (1 μg mL-1 pDNA) as an indicator of the level of endosomal escape after 12 h treatment. Statistical P-values: not significant: NS; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 7]Figures 7A, 7B, 7C, 7D, 7E, 7F, and 7G show sustained expression and extended transgene expression duration of pDNA LNPs with co-delivery of anti-inflammatory siRNA. (Figures 7A-7B) Whole-body bioluminescence imaging of Balb / c mice at different time points after iv administration of a single dose of LNPs containing 25 μg Luc pDNA or 5 μg Luc mRNA per mouse for mRNA LNPs (n=3). (Figures 7C-7E) Whole-body bioluminescence imaging of Balb / c mice at different time points after administration (25 μg Luc pDNA per mouse, 2.5 μg siRNA for each transcription factor per mouse (n=3)). (Figure 7F) Transcription factor levels in treated mice were measured by ELISA 7 days after administration of a single dose. (Figure 7G) A FACScan was used to determine infiltrating inflammatory monocytes (CD45+CD11b+ cells) in the liver after treatment. Data above are presented as mean ± SEM. Statistical P values: not significant: NS; *P<0.05, **P<0.01, ***p<0.001, ****P<0.0001. Unless otherwise indicated, the labels above each group indicate statistical comparisons with the PBS control group. [Figure 8] Figures 8A, 8B, and 8C show the transfection efficiency of LNPs in groups B, D, and E, respectively, prepared using DDAB, DSPC, or 14PA as helper lipids. FACS was used to further evaluate the transfection efficiency of (Figure 8A) DDAB, (Figure 8B) DSPC, and (Figure 8C) 14PA LNPs (1 μg mL-1 pDNA(GFP), 72 h, n=2). Efficiency of transgene expression of GFP as a reporter. Cell metabolic activity was measured by alamarBlue assay (n=4). The formulations were regrouped into four clusters containing eight formulations each based on transgene expression levels. Data are presented as mean ± SD. The percentage of each component in the formulations is shown in pie charts. See Tables 1-6 for the molar percentages of all lipids used in the 32 formulations of each LNP group. [Figure 9]The effect of various formulation parameters on the mean size of LNPs is shown. The mean size and size distribution of the top performing LNPs (top 32 formulations in each LNP group) were measured using dynamic light scattering (DLS). [Figure 10] Survival of Balb / c mice after a single intravenous injection of various LNP formulation clusters is shown. The three most toxic clusters (AII, AIV, DIV) are shown in this survival graph; all other clusters tested did not result in animal death. LNPs were injected at a total pDNA dose of 100 μg (50% Luc + 50% mCherry) per mouse for each cluster (n=2). [Figure 11] Survival rates of Balb / c mice after a single intravenous injection of different LNP formulations are shown. The five most toxic formulations (DI-2, DI-4, FIII-2, FIII-3, FIII-4) are shown in this survival graph; all other formulations tested did not cause any deaths. LNPs were injected at a total pDNA dose of 50 μg (50% Luc + 50% mCherry) per mouse (n=2). [Figure 12] Figure 1 shows the average expression levels of tdTom in different organs 3 days after a single iv injection of LNPs in Ai9 mice. The dose of Cre pDNA was 25 μg per mouse (iv, n=2). Data are presented as mean ± standard deviation. [Figure 13] Figure 1 shows the percentage of tdTom+ cells in major organs 3 days after a single iv injection of LNPs in Ai9 mice. FACS was used to quantify the percentage of tdTom+ cells in each organ (25 μg Cre pDNA per mouse, iv, n=2). Data are presented as mean ± standard deviation. [Figure 14] Z-average size and zeta potential of different LNP formulations in clusters DI and FIII measured by DLS (n=3). Data are presented as mean ± SD. The percentage of each component in the formulation is indicated by the pie chart. See Tables 4 and 6 for the molar percentages used in selected formulations. [Figure 15] Biodistribution in liver 6, 12, and 24 hours after injection of LNPs in different clusters (DI-3, DI-6, DI-8, FIII-1, FIII-7, and FIII-8) is shown. In vivo distribution in liver was measured 6, 12, and 24 hours after injection of LNPs in different clusters at a total pDNA dose of 30 μg per mouse (85% Luc + 15% Cy5-labeled 1216, iv, (n=3). [Figure 16] Figure 1 shows the percentage of apoptotic cells (Zombie Violet- Apotracker Green+ cells) in the liver after a single intravenous injection of one of the selected LNPs. FACS was used to quantify the percentage of Zombie Violet- Apotracker Green+ cells in the liver (25 μg Luc pDNA per mouse, 2.5 μg siRNA for each transcription factor per mouse, iv, n=3). Data are presented as mean ± SEM. [Figure 17]Figures 17A, 17B, 17C, and 17D show the effect of several key formulation parameters, including type of helper lipid, molar ratio of ionic lipid to helper lipid, molar ratio of steroid to PEGylated lipid, total percentage of ionic lipid and helper lipid, and N to P ratio, on in vitro LNP-mediated pDNA delivery using a high-throughput screening platform after 72 hours of incubation (n=2). The transgene expression level for each formulation is displayed using luciferase as a reporter. (Figure 17A) Strip plot of transfection efficiency of 1080 LNPs in B16-F10 melanoma cells grouped by the molar ratio of DLin-MC3-DMA to helper lipid used in the formulation. (Figure 17B) Strip plot of transfection efficiency of 1080 LNPs in B16-F10 melanoma cells grouped by the molar ratio of cholesterol to DMG-PEG2000 used in the formulation. (FIG. 17C) Strip plot of transfection efficiency of 1080 LNPs in B16-F10 melanoma cells grouped by the total percentage of DLin-MC3-DMA and helper lipid used in the formulation. (FIG. 17D) Strip plot of transfection efficiency of 1080 LNPs in B16-F10 melanoma cells grouped by the N and P ratio used in the formulation. [Figure 18]Figures 18A, 18B, 18C, and 18D show the effect of several key formulation parameters, including the molar ratio of ionic lipid to helper lipid, the molar ratio of steroid to PEGylated lipid, the total percentage of ionic lipid and helper lipid, and the N to P ratio, on in vitro LNP-mediated pDNA delivery using a high-throughput screening platform after 72 hours of incubation (n=2). The transgene expression level for each formulation is displayed using luciferase as a reporter. (Figure 18A) Strip plot of transfection efficiency of 1080 LNPs in HepG2 hepatoma cells grouped by the molar ratio of DLin-MC3-DMA to helper lipid used in the formulation. (Figure 18B) Strip plot of transfection efficiency of 1080 LNPs in HepG2 hepatoma cells grouped by the molar ratio of cholesterol to DMG-PEG2000 used in the formulation. (FIG. 18C) Strip plot of transfection efficiency of 1080 LNPs in HepG2 hepatoma cells grouped by the total percentage of DLin-MC3-DMA and helper lipid used in the formulation. (FIG. 18D) Strip plot of transfection efficiency of 1080 LNPs in HepG2 hepatoma cells grouped by the N and P ratio used in the formulation. [Figure 19]Figures 19A, 19B, 19C, and 19D show the effect of several key formulation parameters, including type of helper lipid, molar ratio of ionic lipid to helper lipid, molar ratio of steroid to PEGylated lipid, total percentage of ionic lipid and helper lipid, and N to P ratio, on in vitro LNP-mediated pDNA delivery using a high-throughput screening platform after 72 hours of incubation (n=2). Transgene expression levels for each formulation are shown using luciferase as a reporter. (Figure 19A) Strip plot of transfection efficiency of 1080 LNPs in PC3 human prostate cancer cells grouped by molar ratio of Dlin-MC3-DMA to helper lipid used in the formulation. (Figure 19B) Strip plot of transfection efficiency of 1080 LNPs in PC3 human prostate cancer cells grouped by molar ratio of cholesterol to DMG-PEG2000 used in the formulation. (FIG. 19C) Strip plot of transfection efficiency of 1080 LNPs in PC3 human prostate cancer cells grouped by the total percentage of DLin-MC3-DMA and helper lipid used in the formulation. (FIG. 19D) Strip plot of transfection efficiency of 1080 LNPs in PC3 human prostate cancer cells grouped by the N and P ratio used in the formulation. [Figure 20] Figures 20A and 20B show the in vivo transfection efficiency of selected LNP formulations administered via intraduodenal injection in Balb / c mice. (Figures 20A-B) Liver bioluminescence flux 48 hours after administration of a single dose. The top six formulations (above the dotted line) were selected. Formulation details of these six formulations are shown in Table 7. [Figure 21]Figure 1 shows the transfection efficiency of mRNA LNPs in primary neurons. The transfection efficiency of GFP mRNA LNPs in primary neurons was assessed via flow cytometry after 24 h of transfection (n=3; mRNA 0.5 μg / well). The top eight formulations (A2, A3, A5, B9, C9, D7, D10, F5) were selected for future investigation. The formulation details of these eight formulations are shown in Table 8. [Figure 22]Figures 22a, 22b, 22c, 22d, 22e, 22f, 22g, and 22h show in vitro screening of mRNA lipid nanoparticles for transfection and induction of antigen presentation and maturation in DCs. Figure 22a, Schematic of the screening platform and therapeutic mechanism of mRNA LNP vaccination against solid tumors. 1,080 LNP formulations with different helper lipid and component ratios were evaluated for in vitro transfection efficiency. Top performing formulations were then tested for transfection and antigen presentation on BMDCs, and the in vivo immune responses elicited by selected LNPs were evaluated. LNPs transfect tissue-resident DCs after subcutaneous (sc) injection; or drain into nearby lymph nodes, where they transfect APCs, including DCs. These APCs translate the mRNA and process it into peptides that are presented on major histocompatibility complex molecules on the cell surface. Lipids also trigger activation pathways that promote the expression of costimulatory molecules and release of cytokines. T cells activated by APCs proliferate and migrate to tumor sites to neutralize cancer cells in an antigen-specific manner. DC, dendritic cell; IFN-γ, interferon-γ; MHC, major histocompatibility complex; TAP, transporter involved in antigen processing; TCR, T cell receptor; TNF-α, tumor necrosis factor α. Figure 22b, DC2.4 cells were treated with fLuc mRNA LNPs (1 μg mL-1). Relative luciferase expression after 24 h incubation with fLuc mRNA LNPs is shown in a heat map. Figure 22c, BMDCs were treated with 49 top performing LNPs packaged with mCherry mRNA. Percentage of mCherry+ cells gated on CD11c+ cells after 24 h incubation with mRNA LNPs is shown. Details of the LNP formulations are shown in the pie chart: Dlin-MC3-DMA in red, cholesterol in green, DMG-PEG2000 in yellow, and helper lipids in blue. The top seven formulations, indicated by the red arrows, were selected for further study.df, Antigen presentation (Fig. 22d) was accompanied by maturation levels of BMDCs (Fig. 22e, Fig. 22f) analyzed by flow cytometry after 24 h incubation with seven OVA mRNA-loaded LNPs, PBS, free OVA, or LPS and SIINFEKL peptide. The percentage of SIINFEKL-H-2Kb+ cells (Fig. 22d) that were additionally positive for CD86 (Fig. 22e) or CD40 (Fig. 22f) gated on CD11c+ cells is shown. Fig. 22g, Representative flow cytometry analysis of SIINFEFL-H-2Kb and CD40 expression on BMDCs treated with the three top performing LNPs. Fig. 22h, Secretion levels of IFN-γ, TNF-α and IL-6 in the supernatants of BMDCs after 24 h incubation with three OVA mRNA (mOVA)-loaded LNPs were measured by ELISA and shown in a radar chart. Data represent the mean ± SEM from biologically unrelated samples from a representative experiment of two unrelated experiments (n = 3 for (Fig. 22b-g), n = 4 for (Fig. 22h)). Data were analyzed using one-way ANOVA and Dunnett's multiple comparisons test against the PBS group. *P < 0.05, **P < 0.01, ***p < 0.001. NS, not significant. [Figure 23]Figures 23a, 23b, 23c, 23d, 23e, 23f, 23g, 23h, 23i, 23j, 23k, 23l, and 23m show in vivo evaluation of lymph node cell transfection and immune activation by top LNP formulations. Figure 23a, Schematic of Ai9 mouse model and experiment. Cre recombinase is expressed from exogenously delivered Cre mRNA (mCre) and cleaves LoxP sites flanking the stop sequence in the mouse genome, allowing expression of the fluorescent tdTomato reporter. Figures 23b-c, Ai9 mice were administered the top three LNPs loaded with mCre by intramuscular (im) and subcutaneous injection (10 μg mCre per mouse). Transfection of immune cells in the draining lymph nodes was analyzed by flow cytometry. The percentage of cells positive for tdTomato (Fig. 23b) and CD11c (Fig. 23c) gated on CD45+ cells is shown. Fig. 23d, Timeline for immune activation experiment. C57BL / 6 mice were subcutaneously injected with PBS, free OVA protein, or mOVA-loaded C10, D6, or F5 LNPs (10 μg OVA protein or 10 μg mOVA per injection) three times at 1-week intervals. Mice were sacrificed one week after the last injection, and their splenocytes and lymphocytes were isolated for analysis. Fig. 23e, Fig. 23f, C57BL / 6 mice were subcutaneously injected with PBS, free OVA protein, or mOVA-loaded C10, D6, or F5 LNPs. DC antigen presentation (Fig. 23e) was analyzed through flow cytometry along with maturation level in the draining lymph nodes (Fig. 23f). Positive cells for CD11c and SIINFEKL-H-2Kb (Fig. 23e), and CD86 (Fig. 23f) are shown. Fig. 23g-k, C57BL / 6 mice were administered PBS, free OVA protein, OVA protein mixed with aluminum hydroxide gel (Alhydrogel®) (1:1), or C10, D6, F5 and SM-102 LNPs loaded with mOVA by subcutaneous injection (10 μg OVA protein or 10 μg mOVA per injection).Splenocytes were restimulated in vitro for 6 h with OVA and SIINFEKL peptide (OVA at 100 μg mL-1 and SIINFEKL at 2 μg mL-1) and assessed by flow cytometry and intracellular cytokine staining to determine the percentage of CD8+IFN-γ+ (Figure 23g), CD8+Granzyme B+ (Figure 23h), CD8+TNF-α+ (Figure 23i), and CD4+IFN-γ+ (Figure 23j) cells. Figure 23k, Frequency of IFN-γ producing cells in restimulated splenocytes assessed by ELISPOT. Figure 23l, Percentage of double positive restimulated splenocytes for CD4 and IL-4 assessed by flow cytometry and ICS and indicative of Th2 cells. Figure 23m, Titers of OVA-specific IgG antibodies in serum on day 21 measured by ELISA. Data represent the mean ± SEM from a representative experiment of two independent experiments (n=4 (Fig. 23b, c, e-m) biologically independent samples). Data were analyzed using one-way ANOVA and Dunnett's multiple comparisons test for Fig. 23e-j and l-m, and one-way ANOVA and Turkey's multiple comparisons test for Fig. 23b, c, and k. *P<0.05, **P<0.01, ***P<0.001. NS, not significant. [Figure 24]Figures 24a, 24b, 24c, 24d, 24e, 24f, 24g, 24h, 24i, 24j, 24k, and 24l demonstrate the antitumor efficacy of top mRNA LNP formulations as prophylactic and therapeutic vaccines. Figures 24a-d, Schematic and results of prophylactic vaccination model of OVA-expressing melanoma in C57BL / 6 mice. Mice were subcutaneously injected three times at weekly intervals with PBS, free OVA protein, OVA protein mixed with Alhydrogel® (1:1), or mOVA-loaded C10, D6, F5, or SM-102 LNPs (10 μg mOVA per injection) before subcutaneous inoculation with OVA-expressing melanoma (B16F10-OVA) cells (Figure 24a). Survival curves (Fig. 24B), mean tumor volumes (Fig. 24c), and individual tumor volumes (Fig. 24d) are shown over. Fig. 24e-h, Schematic and results of therapeutic vaccination model of B16F10-OVA in C57BL / 6 mice. Mice were subcutaneously inoculated with B16F10-OVA cells and then subcutaneously injected three times with PBS or mOVA-loaded C10 (10 μg mOVA per injection) at one-week intervals. Two groups received repeated anti-CTLA-4 monoclonal antibody (mAb; 100 μg per intraperitoneal (ip) injection) treatment, alone or in combination with LNP treatment (Fig. 24e). Survival curves (Fig. 24F), mean tumor volumes (Fig. 24g), and individual tumor volumes (Fig. 24h) over time are shown over time. Figure 24i-24l, Schematic and results of therapeutic vaccination model against melanoma-associated antigens for melanoma in C57BL / 6 mice. Mice were inoculated subcutaneously with B16F10 cells and then subcutaneously injected three times at weekly intervals with PBS or C10 LNPs loaded with mRNA encoding Trp2 (mTrp) or Gp100 (mGp100) (10 μg of mRNA per injection). Two groups received anti-CTLA-4 mAb (100 μg per intraperitoneal injection) treatment in combination with LNP treatment (Figure 24i). Survival curves (Figure 24j), mean tumor volumes (Figure 24k), and individual tumor volumes (Figure 24l) over time are shown.Data represent the mean ± SEM from a representative experiment of two independent experiments (n=7 (Fig. 24b-d), n=6 (Fig. 24f-h, i-l) biologically independent samples). Differences between treatment groups were analyzed using one-way ANOVA and Tukey's multiple comparison test. Survival curves were compared using the log-rank Mantel-Cox test, and stacked P values ​​were corrected by the Holm-Sidak method for multiple comparisons with alpha set at 0.05. *P<0.05, **P<0.01, ***P<0.001. NS, not significant; ip, intraperitoneal; αCTLA-4, anti-CTLA-4 monoclonal antibody. [Diagram 25]Figures 25a, 25b, 25c, 25d, 25e, 25f, 25g, 25h, 25i, and 25j show that coordinated attack by T cells and NK cells contributed to long-term protection. Figures 25a-g, Schematic and results of cell depletion experiments in a prophylactic vaccination model of OVA-expressing melanoma in C57BL / 6 mice. Mice were subcutaneously injected three times at weekly intervals with PBS or mOVA-loaded C10 or F5 LNPs (10 μg mOVA per injection) before subcutaneous inoculation with B16F10-OVA cells, and cell depletion antibodies were injected every 4 days (intraperitoneally, 200 μg per mouse) (Figure 25a). Survival curves (Fig. 25b, e), mean tumor volumes (Fig. 25c, f), and individual tumor volumes (Fig. 25d, g) are shown over time (n=6 biologically independent mice per group). Fig. 25h, Tumor infiltrating immune cells including NK cells, T cells, CD8+ T cells, and Treg cells were measured by flow cytometry at day 22 after tumor inoculation (n=6 per group). Fig. 25i, Percentage ratio of CD8+ T cells to CD4+FoxP3+CD25+ Treg cells at day 22 after tumor inoculation (n=6 per group). Fig. 25j, Immunofluorescence analysis of CD3 T cell and NK cell infiltration of tumor sections at day 22 after tumor inoculation. DAPI (blue), CD3 (green), NK1.1 (red), scale bar 50 μm. Data presented as mean ± SEM. Differences between treatment groups were analyzed using one-way ANOVA and Tukey's multiple comparison test. Survival curves were compared using the log-rank Mantel-Cox test, and stacked P values ​​were corrected by the Holm-Sidak method for multiple comparisons with alpha set at 0.05. *P<0.05, **P<0.01, ***p<0.001. NS, not significant. [Figure 26]Figures 26a, 26b, 26c, 26d, 26e, 26f, 26g, 26h, 26i, and 26j show local transfection, intracellular uptake, and endosomal escape of mRNA LNPs. Figure 26a: Schematic of different immune responses induced by mRNA LNPs. Transfected APCs translate, process, and present antigen epitopes on MHC-I molecules to CD8+ T cells, while transfected non-APCs, such as muscle cells, translate and release antigens so that APCs can internalize antigen epitopes on MHC-II molecules and present them to helper T cells. Some exogenous antigens are taken up and presented on MHC-I molecules by the cross-presentation pathway. Figure 26b, Figure 26c, Makeup of transfected cells at the injection site 24 hours after injection of C10, D6, and F5 formulations loaded with GFP mRNA (mGFP). Flow cytometry was used to determine the ratio of non-immune cells to immune cells (Figure 26b) and the relative abundance of each cell type (Figure 26c). (Immune cells (CD45+), epithelial cells (CD326+), endothelial cells (CD31+), myocytes (desmin+), adipocytes (CD45-CD31-CD36+). Figure 26d-Figure 26h, In vitro evaluation of transfection or uptake efficiency by formulations C10, D6, and F5 in C2C12 cells and BMDCs. Flow cytometry was used to determine the ratio of mCherry mRNA-transfected C2C12 cells to transfected BMDCs in in vitro coculture (Figure 26d), the transfection efficiency of LNPs containing fLuc mRNA in pure C2C12 cell cultures (Figure 26e), the transfection efficiency of LNPs containing mCherry mRNA in pure BMDCs (Figure 26f), the transfection efficiency of LNPs containing mGFP in pure C2C12 cells (Figure 26g), and the uptake efficiency of LNPs containing Cy5-labeled mRNA in C2C12 cells (Figure 26h).Lysotracker was used to identify the colocalization of fluorescently labeled lysosomes (Lyso-tracker) and LNPs containing Cy5-labeled mRNA in C2C12 cells in vitro (Figure 26i). Data represent the mean ± standard error from a representative experiment of two independent experiments (n = 4 biologically independent samples for (Figure 26b-Figure 26g)). *P < 0.05, ***P < 0.001, ****P < 0.0001. NS, not significant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description

[0039] The presently disclosed subject matter will now be described in more detail below with reference to the accompanying figures, which show some but not all embodiments of the present invention. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Indeed, many modifications and other embodiments of the presently disclosed subject matter described herein will come to mind to one skilled in the art to which the presently disclosed subject matter pertains having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. It is therefore to be understood that the presently disclosed subject matter is not limited to the specific embodiments disclosed, and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0040] A. Lipid nanoparticles for plasmid DNA delivery to the liver

[0041] In some embodiments, the presently disclosed subject matter provides solid nanoparticles including a nucleic acid payload comprising a steroid, an ionizable cationic lipid, a helper lipid, a PEGylated lipid, and one or more nucleic acids, wherein the nanoparticles comprise: a molar ratio of steroid to PEGylated lipid of between about 10 and about 900; a molar ratio of ionizable cationic lipid to helper lipid of between about 1 and about 200; a total percentage of ionizable lipid and helper lipid of between about 20% and about 80%; and an N to P ratio of between about 2 and about 14.

[0042] In certain embodiments, the presently disclosed subject matter provides solid nanoparticles including a nucleic acid payload comprising a steroid, an ionizable cationic lipid, a helper lipid, a PEGylated lipid, and one or more nucleic acids, wherein the nanoparticles comprise: a molar ratio of steroid to PEGylated lipid of between about 200 and about 900; a molar ratio of ionizable cationic lipid to helper lipid of between about 1 and about 50; a total percentage of ionizable lipid and helper lipid of between about 35% and about 65%; and an N to P ratio of between about 2 and about 14.

[0043] As used herein, the term "steroid" refers to compounds having a core structure that includes four fused rings, including three six-membered cyclohexane rings (annotated as rings A, B, and C) and one five-membered cyclopentane ring (annotated as ring D), as provided in the following structural formula: [ka] .

[0044] The functionality of the steroids can be tailored by varying the substituents on the four ring core, including one or more substituents selected from, for example, alkyl, alkoxyl, hydroxyl, oxo, acyl, and by the oxidation state of the rings. The steroids can also be modified by changing the ring structure, for example by opening one of the rings.

[0045] As used herein, the term "sterol" refers to a subgroup of steroids that have a hydroxyl group at the 3-position of the A ring. Sterols are amphipathic lipids that have a polar hydroxyl group on the A ring, while the remainder of the aliphatic chain is non-polar. Sterols have the following general structure: [ka] .

[0046] In certain embodiments, the steroid is cholestane or a cholestane derivative. In other embodiments, the steroid is a sterol or a sterol derivative. In certain embodiments, the sterol includes cholesterol.

[0047] The term "ionizable cationic lipid" as used herein refers to an ionic lipid that is positively charged at acidic pH and condenses anionic polymers, such as nucleic acids, into lipid nanoparticles. Ionizable cationic lipids are neutral at physiological pH to minimize toxicity. Representative ionizable cationic lipids include, but are not limited to, unsaturated ionic lipids, including DLin-MC3-DMA, OF-02, A6, and A18-Iso5-2DC18; 98N 12 -5, multiterminal ionic lipids including C12-200, cKK-E12, and 9A1P9; ionic polymeric lipids including 7C1 and G0-C14; L319, 304O 13 Biodegradable ionic lipids, including 306O, OF-Deg-Lin, and 306-O12B; i10and FTT5. Other ionizable lipids suitable for use in the solid nanoparticles of the present disclosure include SM-102, ACL-0315, A9, 2,2(8,8) 4C CH3, and LP01. See, for example, Han et al., An ionizable lipid toolbox for RNA delivery, Nature Communications, 12:7233 (2021), which is incorporated herein by reference in its entirety. In certain embodiments, the ionizable cationic lipid includes Dlin-MC3-DMA.

[0048] In some embodiments, the helper lipid is selected from a cationic lipid, a zwitterionic lipid, and an anionic lipid.

[0049] In certain embodiments, the cationic lipid is N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide, 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), O-alkylphosphatidylcholine, 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPD), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), 1,2-diisopropyl-sn-glycero-3-ethylphosphocholine (19:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (20:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (21:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (22:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (23:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (24:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (25:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (26:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (27:0 EPC), 1,2-isopropyl-sn-glycero-3-ethylphosphocholine (28:0 EPC), EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), dimethyldioctadecylammonium (DDAB), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 1,2-distearoyl-3-dimethylammonium propane (18:0 DAP), 1,2-dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-Dioleoyl-3-Dimethylammoniumpropane (DODAP) (18:1 DAP), 1,2-Dimyristoyl-3-trimethylammoniumpropane (14:0 TAP), 1,2-Dipalmitoyl-3-trimethylammoniumpropane (16:0 TAP), 1,2-Stearoyl-3-trimethylammoniumpropane (18:0 TAP), 1,2-Dioleoyl-3-trimethylammoniumpropane (18:1 TAP (DOTAP)), 3β-[N-(N',N'-Dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol-HCl), DC-Cholesterol, N4-Cholesteryl-Spermine (GL67), 1,2-Dioleyloxy-3-dimethylaminopropane (DODMA), dimyristoyltrimethylammoniumpropane (DMTAP), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propane trifluoroacetate (DOSPA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoylcarbamyl-3-dimethylammoniumpropane (DOCDAP), 1,2-dilineoyl-3-dimethylammoniumpropane (DLINDAP), dilauryl (C, 12:0 )trimethylammoniumpropane (DLTAP), dioctadecylamidoglycylspermine (DOGS), DC-Choi (DC Choi), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'- (cholest-5-ene-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA) and N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and combinations and pharma- ceutically acceptable salts thereof.

[0050] In certain embodiments, the cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and dimethyldioctadecylammonium (DDAB). In certain embodiments, the cationic lipid is 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In certain embodiments, the cationic lipid is dimethyldioctadecylammonium (DDAB).

[0051] In some embodiments, the zwitterionic lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), including DSPC50.

[0052] In some embodiments, the anionic lipid comprises a phospholipid. In certain embodiments, the phospholipid is selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0053] In certain embodiments, the solid lipid nanoparticles include a polyethylene glycol lipid conjugate (referred to herein as a "PEGylated lipid" or "PEG lipid"). Exemplary PEGylated lipids include, but are not limited to, N-(carbonylmethoxypolyethylene glycol). n )-1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG n , where n is 350, 500, 750, 1000 or 2000), N-(carbonylmethoxypolyethylene glycol n )-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG n , where n is 350, 500, 750, 1000 or 2000), DSPE-polyglycerol-cyclohexyl-carboxylic acid, DSPE-polyglycerol-2-methylglutaric-carboxylic acid, polyethylene glycol-dimyristylglycerol (DMG-PEG), polyethylene glycol-distearoylglycerol (PEG-DSG), or N-octanoyl-sphingosine-1-{(succinyl[methoxy(polyethylene glycol)2000]}(C8 PEG2000 ceramide). nIn some variations of DSPE-PEGn, where n is 350, 500, 750, 1000, or 2000, the PEG lipid is N-(carbonylmethoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG2,000). In some variations of DSPE-PEGn, where n is 350, 500, 750, 1000, or 2000, the PEG lipid is N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG2,000).

[0054] In certain embodiments, the PEGylated lipid includes dimyristoylglycerol (DMG)-polyethylene glycol (PEG) 2000 (DMG-PEG2000).

[0055] In certain embodiments of the solid nanoparticles of the present disclosure, the steroid comprises cholesterol; the ionizable cationic lipid comprises DLin-MC3-DMA; the PEGylated lipid comprises DMG-PEG2000; the nucleic acid is mRNA; and the helper lipid is selected from 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DSPC, 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0056] In certain embodiments of the solid nanoparticles of the present disclosure, the solid nanoparticles comprise the following: a combined molar percentage of DLin-MC3-DMA and helper lipid ranging from about 20% to about 80%; a weight ratio of cholesterol to DMG-PEG2000 ranging from about 10 to about 500; a weight ratio of DLin-MC3-DMA to helper lipid ranging from about 1 to about 200; and a molar ratio of charged groups on the ionizable lipid to phosphate groups on the mRNA (N / P ratio) ranging from about 4 to about 12.

[0057] In certain embodiments of the solid nanoparticles of the present disclosure, the solid nanoparticles comprise: (a) about 30 mol% DOPE, about 30 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 4; (b) about 7 mol% DSPC, about 70 mol% DLin-MC3-DMA, about 20 mol% cholesterol, about 0.04 mol% DMG-PEG2000, and an N / P ratio of about 4; or (c) about 5 mol% 18PG, about 55 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 12%.

[0058] Representative lipids are disclosed in U.S. Pat. No. 11,229,609, Compositions and methods for organ specific delivery of nucleic acids, by Cheng et al., published January 25, 2022, which is incorporated by reference in its entirety, particularly columns 3-10 and 46-52.

[0059] As used herein, the term "nucleic acid" refers to one or more of the following biomolecules: small interfering ribonucleic acid (siRNA), messenger RNA (mRNA), micro ribonucleic acid (miRNA), primary micro ribonucleic acid (pri-miRNA), messenger ribonucleic acid (mRNA), clustered regularly interspaced short palindromic repeats (CLRs), and / or ribonucleic acid (RIAs). The nucleic acid may include a Crisp RNA (CRISPR)-associated nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a trans-activating CRISPR ribonucleic acid (tracrRNA), a plasmid deoxyribonucleic acid (pDNA), a transfer ribonucleic acid (tRNA), an antisense oligonucleotide (ASO), a guide ribonucleic acid, a double-stranded deoxyribonucleic acid (dsDNA), a single-stranded deoxyribonucleic acid (ssDNA), a single-stranded ribonucleic acid (ssRNA), a double-stranded ribonucleic acid (dsRNA), a protein, a CRSIPR-associated (Cas) protein, or a combination thereof. In certain embodiments, the nucleic acid includes a plasmid DNA (pDNA) or a siRNA. In certain embodiments, the nucleic acid is plasma DNA. In certain embodiments, the nucleic acid includes a siRNA. In even more certain embodiments, the nucleic acid includes a combination of pDNA and siRNA. In certain embodiments, the siRNA is an anti-inflammatory siRNA.

[0060] In some embodiments of the solid nanoparticles, the steroid has a molar ratio of between about 10 and about 900 to the PEGylated lipid, and is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 97 Included are molar ratios of 0, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, and 900.

[0061] In some embodiments of the solid nanoparticles, the ionizable cationic lipid is in a molar ratio to the helper lipid of from about 1 to about 200, including molar ratios of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, and 200.

[0062] In some embodiments, the solid nanoparticles have a total percentage of ionic lipids and helper lipids between about 20% and about 80%, such as about 20%, 21%, 22%, 23%, 25%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%. These include the following total percentages: 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 695, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80%.

[0063] In some embodiments, the solid nanoparticles comprise a P to N ratio of between about 2 and about 14, including P to N ratios of about 2, 2.5, 3, 3.5, 4, 4.5, 5, 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, and 14.

[0064] In some embodiments, the solid nanoparticles have a weight fraction of siRNA in the nucleic acid payload of from about 0 to about 1, including about 0, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, and about 1.

[0065] In some embodiments, the nanoparticles have a size less than about 400 nm, and may be less than about 400 nm, 395 nm, 390 nm, 385 nm, 380 nm, 375 nm, 370 nm, 365 nm, 360 nm, 355 nm, 350 nm, 345 nm, 340 nm, 335 nm, 330 nm, 325 nm, 320 nm, 315 nm, 310 nm, 305 nm, 300 nm, 295 nm, 290 nm, 285 nm, 280 nm, 275 nm, 270 nm, 265 nm, 260 nm, 255 nm, 250 nm, 245 nm, 240 nm, 235 nm, 230 nm, 225 nm, 240 nm, 255 nm, 250 nm, 265 nm, 270 nm, 285 nm, 28 ... Sizes include: 0nm, 215nm, 210nm, 205nm, 200nm, 195nm, 190nm, 185nm, 180nm, 175nm, 170nm, 165nm, 160nm, 155nm, 150nm, 145nm, 140nm, 135nm, 130nm, 125nm, 120nm, 115nm, 110nm, 105nm, 100nm, 95nm, 90nm, 85nm, 80nm, 75nm, 70nm, 65nm, 60nm, 55nm, 50nm, 45nm, 40nm, 35nm, 30nm, 25nm, 20nm, 15nm, 10nm, and 5nm.

[0066] B. Method for delivering lipid nanoparticles containing plasmid DNA to the liver

[0067] In some embodiments, the presently disclosed subject matter provides a method of delivering one or more nucleic acids to the liver of a subject, the method comprising administering to a subject in need of treatment solid nanoparticles of the present disclosure comprising one or more nucleic acids and as described above.

[0068] In some embodiments, the one or more nucleic acids are selected from plasmid DNA (pDNA), mRNA, siRNA, and combinations thereof. In certain embodiments, the one or more nucleic acids are plasmid DNA. In certain embodiments, the one or more nucleic acids are siRNA. In certain embodiments, the one or more nucleic acids include a combination of plasmid DNA (pDNA) and siRNA.

[0069] In certain embodiments, the siRNA comprises an anti-inflammatory siRNA. In such embodiments, the anti-inflammatory siRNA can target a transcription factor selected from signal transduction and activator of transcription (STAT), and nuclear factor kappa light enhancer of activated B cells (NF-κβ). In certain embodiments, the method comprises reducing inflammation-induced gene silencing. In certain embodiments, the duration of expression of the pDNA when administered with the anti-inflammatory siRNA is longer than the duration of expression of the pDNA when administered alone. In certain embodiments, the expression level of the pDNA when administered with the anti-inflammatory siRNA is substantially similar to the expression level of the pDNA when administered alone. In certain embodiments, the method comprises reducing the levels in the liver of one or more signal transduction and activator of transcription (STAT), nuclear factor kappa light enhancer of activated B cells (NF-κβ), one or more infiltrating inflammatory monocytes, and one or more apoptotic cells. In certain embodiments, the one or more infiltrating inflammatory monocytes are CD45 + Cells and CD11b + Selected from cells.

[0070] In some embodiments, the method includes treating one or more diseases or disorders of the liver. In certain embodiments, the one or more diseases or disorders of the liver are selected from genetic liver diseases and inflammatory liver diseases. In certain embodiments, the one or more diseases or disorders of the liver are selected from hemophilia B, hemophilia A, ornithine transcarbamylase (OTC) deficiency, phenylketonuria, acute intermittent porphyria, methylmalonic acidemia, familial hypercholesterolemia, Fabry, MPS type VI (mucopolysaccharidosis type VI), Gangliosidosis GM1, Danon disease, GSDla von Gierke (Glycogen storage disease 1a Von Gierke), Wilson's disease, Crigler-Najjar, primary hyperoxaluria type 1, and combinations thereof.

[0071] In certain embodiments, the method for delivering one or more nucleic acids to the liver of a subject is selected from intravenous (iv) injection, oral, subcutaneous, and inhalation delivery.

[0072] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a disorder and / or symptoms associated therewith. It will be understood that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated, although this is not precluded.

[0073] The "subject" treated by many embodiments of the disclosed methods is desirably a human subject, although it should be understood that the methods described herein are effective for all vertebrate species intended to be included in the term "subject." Thus, "subject" includes human subjects for medical purposes, such as treatment of an existing condition or disease, or prophylactic treatment to prevent the onset of a condition or disease, or animal subjects for medical, veterinary, or development purposes. Suitable animal subjects include mammals, including, but are not limited to, primates, such as humans, monkeys, apes, and the like; bovines, such as cattle, bulls, and the like; ovines, such as sheep and the like; caprines, such as goats and the like; porcines, such as pigs, boars, and the like; equines, such as horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, such as mice, rats, and the like. The animals can be transgenic animals. In some embodiments, the subject is a human, including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Additionally, a "subject" can include a patient suffering from or suspected of suffering from a condition or disease. Thus, the terms "subject" and "patient" are used interchangeably herein. The term "subject" may refer to an organism, tissue, cell, or collection of cells from a subject.

[0074] C. Methods for Preparing Lipid Nanoparticles for Plasmid DNA Delivery to the Liver

[0075] In some embodiments, the presently disclosed subject matter provides a method for preparing a solid nanoparticle of the present disclosure, the method comprising: (a) preparing an organic phase by dissolving a mixture of steroid, ionizable cationic lipid, helper lipid, and PEGylated lipid in a predetermined molar ratio in a polar protic solvent; (b) preparing an aqueous phase by dissolving one or more nucleic acids in an aqueous buffer; and (c) mixing the organic phase and the aqueous phase to form solid nanoparticles. Includes.

[0076] In some embodiments, the polar protic solvent comprises a branched or straight chain C1-C4 alcohol, including C1, C2, C3, C4 alcohols. Representative C1-C4 alcohols include, but are not limited to, methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, and tert-butanol. In certain embodiments, the polar, protic solvent is ethanol.

[0077] In certain embodiments, the aqueous buffer includes a magnesium acetate buffer.

[0078] In some embodiments, the method further comprises mixing the organic and aqueous phases in a flash nanocomplexation (FNC) device. As used herein, the term "flash nanocomplexation" refers to a method employing two or more impinging jets in a mixing chamber. As provided in International PCT Patent Application Publication No. WO2021252715 for Axisymmetric Confined Impinging Jet Mixer to Mittel et al., published on December 16, 2021, and incorporated herein by reference in its entirety, these devices include: (a) flowing a first stream containing one or more water-soluble polycationic polymers into a confined chamber; (b) flowing a second stream containing one or more water-soluble polyanionic polymers, such as plasma DNA or siRNA, into a confined chamber; and (c) impinging the first and second streams into the confined chamber, thereby generating one or more water-soluble polycationic polymers and one or more water-soluble polyanionic polymers to undergo a polyelectrolyte complexation process that continuously produces PEC nanoparticles. These types of devices, which use two or more impinging jets in a confined mixing chamber, are referred to in the prior art as confined impinging jet (CIJ) mixers. Turbulence-induced mixing can be achieved by T-connectors, Tesla mixers, herringbone mixers, coaxial jet mixers, confined impinging jet mixers (CIJMs), and multi-inlet vortex mixers (MIVMs).See, for example, International PCT Patent Application Publication No. WO2020223323, published on November 5, 2020, to Mao et al., for Compositionally Defined Plasmid DNA / Polycation Nanoparticles And Methods For Making The Same; U.S. Patent Application Publication No. 20170042829, published on February 16, 2017, to Mao et al., for Methods of Preparing Polyelectrolyte Complex Nanoparticles; and U.S. Patent No. 10,441,549, published on October 15, 2019, to Mao et al., for Methods of Preparing Polyelectrolyte Complex Nanoparticles, the contents of each of which are incorporated herein by reference in their entirety.

[0079] In some embodiments, the method further comprises mixing the organic phase and the aqueous phase in a ratio of about 3:1, including a ratio of about 3:1, a ratio of about 2.5:1, a ratio of about 2.0:1, a ratio of about 1.5:1, and a ratio of about 1:1.

[0080] In some embodiments, the method further comprises dialysing the solid nanoparticles against deionized water.

[0081] D. Methods of Stimulating Th1 and / or Th2 Responses

[0082] In some embodiments, the presently disclosed subject matter provides a method of stimulating or inducing a type 1 T helper (Th1) and / or type 2 T helper (Th2) response in vivo, the method comprising administering a solid nanoparticle of the present disclosure.

[0083] In certain embodiments, the steroid comprises cholesterol; the ionizable cationic lipid comprises DLin-MC3-DMA; the PEGylated lipid comprises DMG-PEG2000; the nucleic acid comprises mRNA; and the helper lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DSPC, 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

[0084] In certain embodiments, the solid nanoparticles comprise the following: a combined molar percentage of DLin-MC3-DMA and helper lipid ranging from about 20% to about 80%; a weight ratio of cholesterol to DMG-PEG2000 ranging from about 10 to about 500; a weight ratio of DLin-MC3-DMA to helper lipid ranging from about 1 to about 200; and a molar ratio of charged groups on the ionizable lipid to phosphate groups on the mRNA (N / P ratio) ranging from about 4 to about 12.

[0085] In certain embodiments, the solid nanoparticles comprise: (a) about 30 mol% DOPE, about 30 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 4; (b) about 7 mol% DSPC, about 70 mol% DLin-MC3-DMA, about 20 mol% cholesterol, about 0.04 mol% DMG-PEG2000, and an N / P ratio of about 4; or (c) about 5 mol% 18PG, about 55 mol% DLin-MC3-DMA, about 40 mol% cholesterol, about 0.40 mol% DMG-PEG2000, and an N / P ratio of about 12.

[0086] In certain embodiments, the method induces an immune response in only Th1, only Th2, or both Th1 and Th2.

[0087] E. Methods of Treating a Disease or Disorder

[0088] In other embodiments, the presently disclosed subject matter provides a method of treating a disease, disorder, or condition in a subject, the method comprising administering to a subject in need of treatment a therapeutically effective amount of the solid nanoparticles of the present disclosure.

[0089] In certain embodiments, the disease is selected from cancer or infectious disease. In particular embodiments, the cancer is selected from basal cell carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal carcinoma, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, malignant pleural mesothelioma, Merkel cell carcinoma, metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer, squamous cell carcinoma, and urothelial carcinoma.

[0090] In certain embodiments, the infection comprises a viral infection, hi particular embodiments, the viral infection is selected from a coronavirus infection, a Zika virus infection, influenza, a flavivirus infection, and a human immunodeficiency virus (HIV) infection.

[0091] In other embodiments, the method further comprises administering the solid nanoparticles together with one or more immune checkpoint inhibitors. In certain embodiments, the immune checkpoint inhibitors are selected from CTLA-4 inhibitors, PD-1 inhibitors, and PD-L1 inhibitors. In certain embodiments, the one or more immune checkpoint inhibitors are selected from ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

[0092] The term "combination" is used in its broadest sense and means that a subject is administered at least two agents, more specifically an agent described herein and at least one other therapeutic agent. More specifically, the term "in combination" refers to administering two (or more) active agents simultaneously, for example, for the treatment of a single disease state. As used herein, the active agents may be combined and administered in a single dosage form, administered simultaneously in separate dosage forms, or administered as separate dosage forms that are administered alternately or sequentially on the same or different days. In one embodiment of the subject matter of the present disclosure, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., where it is desired to vary the amount of one but not the other). The single dosage form may include an additional active agent for treating a disease state.

[0093] Additionally, the agents described herein can be administered alone or in combination with adjuvants that enhance the stability of the compounds, ease administration of pharmaceutical compositions containing them in certain embodiments, provide enhanced solubility or dispersion, enhance inhibitory activity, provide adjunctive therapy, and the like, and include other active ingredients. Advantageously, such combination therapies utilize lower dosages of conventional therapeutic agents, thus avoiding possible toxic and adverse side effects incurred when those agents are used as monotherapies.

[0094] The timing of administering the agent described herein and at least one additional therapeutic agent can be varied, as long as the beneficial effect of the combination of these agents is achieved.Therefore, the phrase "in combination" refers to administering the agent described herein and at least one additional therapeutic agent simultaneously, sequentially, or a combination thereof.Thus, the subject who is administered the combination of the agent described herein and at least one additional therapeutic agent can receive one agent and at least one additional therapeutic agent simultaneously (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), as long as the combined effect of both agents is achieved in the subject.

[0095] When administered sequentially, the agents can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or more of each other. In other embodiments, the sequentially administered agents can be administered within 1, 5, 10, 15, 20 days or more of each other. When the agents described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions each containing either an agent or at least one additional therapeutic agent, or can be administered to the subject as a single pharmaceutical composition containing both agents.

[0096] When administered in combination, the effective concentration of each agent to elicit a particular biological response may be lower than the effective concentration of each agent when administered alone, thereby allowing for a reduction in the dosage of one or more agents relative to the dosage that would be required if the agent were administered as a single agent. The effects of the various agents may be, but are not necessarily, additive or synergistic. The agents may be administered at various times.

[0097] In some embodiments, two or more agents can have a synergistic effect when administered in combination. As used herein, the terms "synergistic effect," "synergistic," "synergistically," and derivatives thereof, such as "synergistic effect" or "synergistic combination" or "synergistic composition," refer to a situation in which the biological activity of a combination of an agent described herein thereunder and at least one additional therapeutic agent is greater than the sum of the biological activities of each agent when administered individually.

[0098] Synergy can be expressed in terms of the "synergy index (SI)", which is usually calculated by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), as follows: Q a / Q A +Q b / Q B = Synergy Index (SI) and During the ceremony: Q A is the concentration of component A that acts alone and produces the endpoint relative to component A; Q a is the concentration of component A in the mixture that produced the endpoint; Q B is the concentration of component B that acts alone and in relation to component B produces an endpoint; and Q b is the concentration of component B in the mixture that produced the endpoint.

[0099] In general, Q a / Q A and Q b / Q BWhen the sum is greater than 1, antagonism is indicated. When the sum is equal to 1, additivity is indicated. When the sum is less than 1, synergy is demonstrated. The lower the SI, the greater the synergistic effect exhibited by that particular mixture. Thus, a "synergistic combination" has greater activity than can be expected based on the observed activity of the individual components when used alone. Furthermore, a "synergistically effective amount" of a component refers to, for example, the amount of the component required to provide a synergistic effect on another therapeutic agent present in the composition.

[0100] F. Vaccines

[0101] In other embodiments, the presently disclosed subject matter provides a vaccine comprising the solid nanoparticles disclosed herein, hi certain embodiments, the vaccine is a cancer vaccine or an anti-viral vaccine.

[0102] Following long-standing patent law practice, the terms "a," "an," and "the" when used in this application (including the claims) refer to "one or more." Thus, for example, the term "an" includes a plurality of objects unless the context clearly indicates to the contrary (e.g., that the object is a plural object).

[0103] Throughout this specification and the claims, the terms "comprise," "comprises," and "comprising" are used in a non-exclusive sense, unless the context otherwise requires. Similarly, the term "include" and its grammatical variations are intended to be open-ended, and the recitation of items in a list does not exclude other similar items that may be substituted for or in addition to the listed items.

[0104] For purposes of this specification and the appended claims, unless otherwise indicated, all numbers expressing quantities, sizes, dimensions, ratios, shapes, formulations, parameters, percentages, quantities, properties, and other numerical values ​​used in this specification and the claims are understood to be modified in all instances by the term "about," even if the term "about" would not be expressly stated in conjunction with the value, amount, or range. Thus, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are not, and need not be, precise, but may be approximated and / or made larger or smaller as desired to reflect tolerances, conversion factors, rounding, measurement errors and the like, as well as other factors known to those of skill in the art, depending upon the desired properties sought to be obtained by the subject matter of the present disclosure. For example, the term "about," when referring to a value, can in some embodiments be meant to encompass the following variations: in some embodiments, ±100%, in some embodiments, ±50%, in some embodiments, ±20%, in some embodiments, ±10%, in some embodiments, ±5%, in some embodiments, ±1%, in some embodiments, ±0.5%, and in some embodiments, ±0.1% from the specified amount, as such variations are appropriate for performing the disclosed methods or employing the disclosed compositions.

[0105] Furthermore, the term "about," when used in connection with a number or range of numbers greater than or equal to one, refers to all such numbers and should be understood to include all numbers in a range and to modify that range by extending the boundaries above and below the numerical values ​​set forth. Recitation of numerical ranges by endpoints includes all numbers subsumed within that range, e.g., integers, fractions thereof, (e.g., recitation of 1 through 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like), and any range within that range. EXAMPLES

[0106] The following examples are included to provide guidance to those of ordinary skill in the art for carrying out representative embodiments of the subject matter of the present disclosure. In light of this disclosure and the general level of skill in the art, the skilled artisan will recognize that the following examples are intended to be exemplary only, and that a vast number of changes, modifications, and variations can be adopted without departing from the scope of the subject matter of the present disclosure. The following synthetic descriptions and specific examples are for illustrative purposes only, and should not be construed as limiting in any way the making of the disclosed compounds by other methods.

[0107] Example 1

[0108] Multi-step screening and composition optimization of lipid nanoparticles for long-term liver-targeted DNA delivery

[0109] 1.1 Overview

[0110] Lipid nanoparticles (LNPs) hold great potential as effective non-viral vectors for gene therapy. Plasmid DNA (pDNA) delivery may result in extended transgene expression compared to mRNA-based technologies, yet systematic investigations on LNP compositions for pDNA delivery are lacking. Here, we provide a multi-step screening platform to identify formulations optimized for liver-targeted transgene expression. To achieve this, we analyzed the role of different helper lipids and component ratios in vitro and in vivo. Compared to mRNA LNPs, the identified formulations were successfully delivered and mediated long-term expression. This platform efficiently selects valid candidates from a library of over 1,000 formulations by addressing different physiological barriers stepwise. Moreover, the expression duration was significantly extended by using a pDNA / siRNA co-delivery approach targeting transcription factors that control inflammatory responses, which highlights the benefits of extended expression profiles using pDNA and provides new opportunities for pDNA-based medical applications.

[0111] 1.2 Scope

[0112] This example provides a multi-step screening platform to systematically test and analyze the liver-targeted transfection efficiency of 1,080 LNP formulations with different helper lipid and component ratios in vitro and in vivo (Figure 1). Overall, a cohort of formulations achieving the highest levels of in vitro transfection efficiency was first identified via high-throughput screening. Motivated by the pooled diagnostic testing methodology used widely during the COVID-19 pandemic, we used a cluster-mode screening approach in the initial in vivo screening phase in eight groups. These clusters were initially screened via intrahepatic injection to assess local toxicity and transgene expression levels. Clusters with minimal cytotoxicity and highest transfection efficiency were then selected for intravenous (iv) injection testing; and formulations within the clusters that showed optimal liver transfection were further evaluated for iv injection individually. This multi-step compositional screening platform was used to identify the most efficient pDNA LNP formulations from the designed library for liver-targeted transfection via iv administration. The transgene expression levels and duration of the optimized pDNA LNPs were also compared with the widely used pDNA / PEI nanoparticles and mRNA LNPs. Hu et al., 2019. To further understand the rate-limiting steps of the in vivo gene delivery process of pDNA LNPs, the in vivo distribution profiles, cellular uptake levels, and lysosomal escape capabilities of the top performing formulations were examined and compared with less effective formulations that share similar properties.

[0113] Another challenge that must be overcome to fully realize the potential of LNP-mediated pDNA delivery is immune-mediated silencing of transgenes. Yew et al., 2002; Ballas et al., 1996; Krieg et al., 1995; Yew et al., 2000; Sparwasser et al., 1998; Hartmann and Krieg, 1999. To extend expression duration and reduce immune responses, several approaches have been explored, including sequence modifications to reduce CpG island density and optimization of delivery carriers. Handumrongkul et al., 2019; Yew et al., 2002.

[0114] To further improve the delivery efficiency of the top formulations, we describe a novel pDNA and siRNA co-delivery strategy targeting key transcription factors that regulate inflammatory response pathways to reduce inflammation-induced gene silencing. Using the optimized LNP formulations, we investigated the effect of co-delivery of pDNA and siRNAs against signal transduction and activator of transcription (STAT), Pfitzner et al., 2004, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), Liu et al., 2017; Lawrence, 2009; Taniguchi and Karin, 2018, on the level and duration of transgene expression after iv administration.

[0115] 1.3 Results

[0116] 1.3.1 Design of LNP libraries for screening of LNP-mediated pDNA delivery

[0117] DLin-MC3-DMA was chosen as the ionic lipid, and DMG-PEG2000 was used as the PEGylated lipid. Six helper phospholipids previously used in experimental or FDA-approved LNP formulations were chosen to represent a range of charges for testing: cationic 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and dimethyldioctadecylammonium (DDAB); zwitterionic 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DSPC; anionic 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (18PG). Hou et al., 2021; Cheng et al., 2020; Cheng and Lee, 2016.

[0118] Using DLin-MC3-DMA, cholesterol, DMG-PEG2000, and one of six helper lipids, an initial library of 1,080 LNP formulations was designed by varying the following parameters: (1) ratio of DLin-MC3-DMA to helper lipid ranging from 1 to 200; (2) ratio of cholesterol to DMG-PEG2000 ranging from 10 to 500; (3) combined percentage of DLin-MC3-DMA and helper lipid ranging from 20% to 80%; and (4) N / P ratio ranging from 4 to 12. This parameter design provided a sufficiently diverse library of LNP formulations with which LNP-mediated pDNA delivery could be programmatically tested.

[0119] To select LNP formulations with strong liver-specific transgene expression, we first evaluated pDNA delivery efficiency of the entire library using firefly luciferase pDNA and measured luciferase protein expression in HepG2 cells (human liver cancer cell line) (Figure 2A). When immobilizing helper lipids, gene expression levels varied significantly depending on the above four parameters in the LNP formulations. Among 1,080 LNP formulations, the top-performing 32 LNPs for each helper lipid group are shown in Figure 2B and Tables 1-6. We then examined the cytotoxicity and evaluated the transfection efficiency of the top 32 formulations by flow cytometry analysis. The results shown in Figures 2C-E and 8 confirmed the high in vitro transfection efficiency and good biocompatibility of these LNPs.

[0120] The average size and size distribution of the top performing LNPs were further measured using dynamic light scattering (DLS) (FIG. 2F). The results indicated that most of the LNPs were less than 400 nm in size (approximately 73.9%), and the percentage of smaller LNPs (less than 400 nm) was higher (approximately 80%) for cationic helper lipids compared to others (e.g., approximately 68% for anionic helper lipids) (FIG. 2G). Upon detailed analysis, several correlations between the average size and formulation parameters were observed: (a) as the N / P ratio increased from 4 to 12, the average size became more uniform (i.e., the standard deviation was lower) (Figure 9A); (b) as the cholesterol content (mol percentage) increased from approximately 20% to approximately 80%, the average LNP size increased, but there was no significant change in the polydispersity index (PDI) (Figure 9B); and (c) as the DMG-PEG2000 content (mol percentage) increased from approximately 0.03% to approximately 5.5%, the LNP size became more uniform (Figure 9C).

[0121] 1.3.2 LNP-mediated intrahepatic pDNA delivery via cluster mode in vivo screening

[0122] The in vivo transfection efficiency of LNPs may differ from that of traditional in vitro assay screens due to differences in in vivo and in vitro settings and delivery barriers. To evaluate their local in vivo tissue transfection efficiency, LNPs that showed the highest range of in vitro transfection efficiency were tested by intrahepatic injection (Figure 3A). As previously mentioned, this in vivo screening process used a cluster mode screening method, which significantly reduced the number of animals required, time, and cost.

[0123] The top performing LNP formulations were first grouped into four clusters per helper lipid (24 clusters in total and 8 formulations per cluster) based on in vitro transfection efficiency (Figure 1C-E and Figure 8). The effect of each cluster was examined by delivering a combination of two plasmids, luciferase (Luc) (50%) and mCherry (50%) pDNA, via intrahepatic injection at a dose of 3 μg total pDNA per mouse. Surprisingly, clusters with high transfection efficiency in vitro were not necessarily the top performing clusters in vivo; and this finding applied both between clusters containing the same helper lipid and between all 24 clusters (Figure 3B-D). For example, cluster AIV, which contains the eight DOTAP preparations with the lowest in vitro transfection efficiency in the DOTAP group, produced a 17.2-fold higher average bioluminescence signal (Luc expression) than cluster A1 (composed of the eight DOTAP preparations with top performance in vitro) (Figure 3D). Furthermore, clusters that showed moderate efficiency in vitro could have strong transfection efficiency in vivo. As an example, cluster FIII showed a surprisingly high transfection efficiency in contrast to its in vitro performance. In addition, the charge of the helper lipid significantly affected the transfection efficiency; cationic lipids such as DOTAP exerted a stronger effect than the others, especially compared to 14PA, the least effective anionic lipid. However, although DOTAP and DDAB are both cationic lipids, the local transfection of DDAB clusters (such as BI, BII, BIV, etc.) in the liver was low. Similarly, most of the clusters composed of anionic helper lipids showed limited local transfection in the liver, whereas the 18PG group had a unique cluster (FIII) that achieved relatively high transfection efficiency. Similarly, the DOPE and DSPC clusters had high overall transfection efficiency, whereas clusters CIII and DIII showed much lower efficiency.No significant luciferase expression was detected in other organs based on gross image analysis.

[0124] To obtain an initial estimate of the cell types transfected by the top performing 12 clusters (shown above the dashed line in Figure 3B), the mCherry expression levels in various cell types in the liver were quantified using flow cytometry (Figure 3E). After a single intrahepatic injection, in all the top 12 clusters, ~50% of the transfected cells in the liver were hepatocytes, followed by immune cells. Notably, for clusters AI and AII, the transfection efficiency in hepatocytes reached as high as 73.9% and 79.5%, respectively. This result indicated that these clusters contained LNP preparations that may have strong liver-specific transfection. Based on the IVIS results (Figure 3B and Figure 3D), the top 12 LNPs clusters (96 preparations in total) were successfully selected as candidate clusters for further evaluation of their stability in the blood circulation and tissue-specific transfection efficiency after systemic delivery.

[0125] 1.3.3 LNP-mediated liver-specific pDNA delivery via cluster mode testing

[0126] Next, the performance of the 12 clusters that showed the highest transgene expression levels in the liver was examined by the iv route. The same payload, luciferase (Luc) (50%) and mCherry (50%) pDNA, was encapsulated in these LNPs, which were administered iv at a total pDNA dose of 100 μg per mouse. Three of the 12 clusters tested (All, AIV, and DIV) showed significant toxicity after iv administration and were excluded from further evaluation (Figure 10). Five clusters (A1, CI, CII, DI, and FIII) were the most efficient for liver-specific transgene expression (Figure 4A-Figure 4D). Compared with the other clusters, these five clusters showed an average of 2-3 orders of magnitude higher luciferase expression in the liver at 12 hours after iv administration measured by IVIS imaging. For example, cluster DI was 660-fold higher than that of cluster DII. For these top 5 LNP clusters, delivery to specific cell types in the liver was further quantified using flow cytometry to detect mCherry expression, which revealed that approximately 40% of transfected cells in the liver were hepatocytes, and approximately 7% of total hepatocytes in the liver were successfully transfected (Figure 4E and Figure 4F).

[0127] Next, the transgene expression levels of the top five clusters were evaluated in other organs, including spleen, lung, kidney, and heart (Figure 4G). Based on the relative Luc expression in each organ, two clusters, DI and FIII, showed high liver-specific transfection efficiency, with 89.6% of the bioluminescence between organs coming from the liver for DI and 93.0% for FIII (Figure 4H). In addition, it was observed that the transgene expression levels in the spleen were higher in clusters CI (45.2%) and CII (30.0%). The percentage of transfected cells in the three major organs (liver, spleen, and lung) was further evaluated by flow cytometry, and in all five clusters, roughly 10% of the cells in the liver were transfected based on mCherry expression (Figure 4I). Based on these data, clusters DI and FIII were selected for further characterization.

[0128] 1.3.4 Formulations for Liver-Specific pDNA Delivery

[0129] The transfection efficiency of 16 individual LNP formulations within the DI and FIII clusters was further examined after iv injection at a total pDNA dose of 50 μg per mouse using the same Luc / mCherry combination (50 / 50) payload. Five of the 16 formulations (DI-2, DI-4, FIII-2, FIII-3, and FIII-4) exhibited high toxicity after iv administration and were excluded from further evaluation (Figure 11). Based on the IVIS results shown in Figures 5A-5D, four individual formulations (DI-3, DI-8, FIII-7, and FIII-8) demonstrated the highest levels of Luc expression in the liver. The best performing formulation, FIII-7, demonstrated 300-fold higher Luc expression than another formulation, FIII-5, within the same cluster. Transgene expression levels in other major organs mediated by the top four formulations were also evaluated (Figure 5E). Of the total bioluminescence in various organs, 73.9% occurred in the liver for FIII-7 and 60.8% in DI-8, indicating exclusive liver-specific transgene expression in both preparations (Figure 5F). Therefore, these top four preparations (DI-3, DI-8, FIII-7, and FIII-8) were taken forward for further testing using orthogonal assays to measure liver-specific transgene expression.

[0130] 1.3.5. Validation of Top Formulations for Liver-Specific pDNA Delivery

[0131] To further validate the efficiency of the top four LNP preparations in mediating liver-specific gene delivery, genetically engineered tdTom reporter mice (Ai9 mice) were used that contain a LoxP-flanked STOP cassette that prevents the expression of tdTom protein. In this mouse model, gene-edited cells can be detected as a result of Cre expression (Figure 5G). When Cre recombinase is introduced into reporter mouse cells, it recombines DNA at the LoxP sites such that the STOP cassette is excised, which allows the expression of fluorescent tdTom. Four preparations (DI-3, DI-8, FIII-7, and FIII-8) were used to deliver Cre recombinase pDNA after iv injection (25 μg pDNA / mouse). At 3 days after injection, high tdTom signals were detected in the liver (Figure 5H); and tdTom-positive cells were easily observed using confocal imaging of tissue sections (Figure 5I). Fluorescent signals were also observed in other organs (Figure 5J, Figure S5), but approximately 60% of the total tdTom expression across imaged organs was from the liver for all four LNPs (Figure 5K). We used flow cytometry to further quantify the percentage of gene-edited cells in the liver and found that approximately 20% of cells were successfully edited by treatment with FIII-8 (Figure 5L, Figure S13). Based on the high transfection efficiency and high biocompatibility, the top four LNPs may be applicable for liver-targeted gene therapy with systemic delivery.

[0132] 1.3.6 In vivo distribution, cellular uptake and endosomal escape levels of top performing formulations

[0133] Although the top four LNP formulations were identified using a multi-step screening platform, the mechanism remains unclear. Without wishing to be bound by a particular theory, the enhanced liver-targeted transfection is a result of: (1) tissue-specific in vivo distribution of LNPs, (2) different cellular uptake profiles of LNPs after distribution to local tissues, and (3) endosomal escape or differential DNA release capabilities of LNPs among formulations with similar in vivo distribution and cellular uptake levels.

[0134] To test these hypotheses, we used Cy5-labeled pDNA to evaluate the in vivo distribution of the four top-performing LNPs at 6, 12, and 24 hours after iv injection. For comparison, we included two LNP formulations (DI-6 and FIII-1) that are less effective than the top performers but have similar properties (size and zeta potential) (Figure 14). IVIS imaging showed that all six selected formulations had similar in vivo distribution profiles at all time points, regardless of transfection performance (Figure 6A, Figure 15). In all cases, approximately 60% of the LNPs were distributed to the liver at 6 hours after iv administration. In addition, similar levels of uptake by hepatocytes were observed for all six formulations, and flow cytometry evaluation indicated that the high transfection by the top formulations was not due to differences at the level of in vivo distribution or cellular uptake (Figure 6B).

[0135] To confirm that the transfection efficiency of LNPs is not related to in vivo distribution, the transfection efficiency of six LNPs was checked by intrahepatic injection of the same dose of six LNPs. The results in Figure 6C and Figure 6D show that the local transfection efficiency differed significantly even though the same dose was delivered to the liver. Compared with DI-6 and FIII-1, the top four formulations indeed provided higher transfection efficiency. Thus, regardless of the delivery route, the transfection efficiency was not strictly related to the in vivo distribution.

[0136] To confirm that endosomal escape and / or DNA release enhance the transfection efficiency of LNP preparations, primary mouse hepatocytes were isolated and transfected with six LNPs (DI-3, DI-8, FIII-7, FIII-8, DI-6, and FIII-1). To simulate the in vivo situation, LNPs were incubated with fresh mouse serum at a 2:1 volume ratio (LNP / serum) for 30 min at 37 °C before dosing with cells. All six LNPs, with or without preincubation with mouse serum, showed similar uptake levels, which are consistent with the observed in vivo cellular uptake levels (Figure 6E). Preincubation with mouse serum did not significantly affect the cellular uptake levels, but significantly improved the transfection efficiency of the best four LNPs. FIII-7 increased the percentage of GFP-positive cells by 12-fold after serum preincubation (Figure 6F and Figure 6G), whereas no significant changes were observed in the transfection efficiency of DI-6 and FIII-1 compared to the dramatic increase for the top four formulations. Furthermore, the endosomal escape ability of selected formulations was examined by quantitative Cellomics high-content analysis on an established B16-Gal8-GFP cell line (Karlsson et al., 2020; Hu et al., 2021). The results shown in Figure 6H indicate that the top four formulations (DI-3, DI-8, FIII-7, and FIII-8) have relatively high endosomal escape ability.

[0137] The above results indicated that LNPs with similar size and zeta potential produced similar in vivo distribution and cellular uptake profiles after iv injection; the variable endosomal escape ability of different LNP formulations is likely the determining factor for the difference in transfection efficiency. Furthermore, the significant differences observed between the groups incubated with or without serum suggested that the discrepancy between in vivo and in vitro experiments may be closely related to serum-mediated opsonization of LNPs.

[0138] 1.3.7 Prolonged duration of transgene expression in LNPs by co-delivery of anti-inflammatory siRNAs

[0139] The duration of expression in the liver of BALB / c mice was monitored after iv injection of the top four formulations (DI-3, DI-8, FIII-7, and FIII-8) (Figure 7A and Figure 7B). The initial expression levels of the four formulations were consistent with the data above, and expression was maintained at similar levels for approximately 4 days before declining over days 3-7. A control group was also tested in which Luc mRNA was loaded using the FIII-7 LNP formulation (5 μg mRNA per mouse, iv). Initial expression in the liver with the mRNA LNPs was comparable to that with pDNA LNPs (25 μg pDNA / mouse) on day 1, but expression levels declined 10-fold on day 2 and more than 300-fold on day 4. The polycationic carrier Polyplus in vivo-jetPEI used to generate pDNA / PEI nanoparticles (PEI NPs) was also tested for comparison. The majority of Luc expression levels mediated by PEI NPs were found in the lungs, and transgene expression in the liver was much lower (approximately 2.4%) than FIII-7 pDNA LNPs at day 1 (Hu et al., 2019). Thus, pDNA LNPs provided significantly longer transgene expression than any of the comparators tested.

[0140] To further extend pDNA expression, several methods have been explored to reduce undesired innate immune activation and host toxicity, such as sequence modification (CpG-depleted pDNA) and optimization of delivery vectors (Handumrongkul et al., 2019; Yew et al., 2002). Here, we tested the effect of co-delivering pDNA and anti-inflammatory siRNAs (against signal transduction and activator of transcription (STAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ)) with pDNA to extend expression duration (Pfitzner et al., 2004; Liu et al., 2017; Taniguchi and Karin, 2018; Salas et al., 2020; Jay and Eric, 2009).

[0141] In these experiments, 2.5 μg siRNA / mouse was used for each transcription factor. As shown in Figure 7C-7E, when STAT and NF-κβ were co-delivered, the duration of expression mediated by FIII-7 LNPs was extended from 5 to 10 days without significant changes in expression levels. In addition, the elevated initial expression levels in livers administered with FIII-7(STAT+NF-κβ) LNPs and FIII-7(STAT) LNPs were 3.4- and 2.2-fold higher than FIII-7 LNPs alone on day 2 (Figure 7E). On day 7 after administration, STAT1 and NF-κβ2 levels in livers were examined by ELISA. As shown in Figure 7F, elevated levels of both inflammatory transcription factors were observed after iv injection of FIII-7 LNPs; and also, by coupling with anti-inflammatory siRNA, the levels of the transcription factors were significantly reduced. In the FIII-7 (STAT+NF-κβ) LNP group, the levels of NF-κβ2 and STAT1 were reduced by approximately 17.6% and 24.5%, respectively, compared to the FIII-7 LNPs. In addition, the liver-infiltrating inflammatory monocytes (CD45 + CD11b +We also observed a reduction in the levels of mitotic cells (cells) and apoptotic cells (Figure 7G, Figure 16). Zigmond et al., 2014. Thus, these combined effects were sufficient to significantly increase the level and duration of transgene expression.

[0142] 1.4 Discussion

[0143] Owing to the success of COVID-19 mRNA vaccines, the application of LNPs as carriers for gene delivery has progressed by leaps and bounds in the past few years. The safety and translatability of LNPs in biological studies have been demonstrated, making it highly attractive for the field of gene therapy. However, to extend this to other therapeutic areas, LNP formulations need to be systematically screened and optimized based on requirements such as specific target cell and tissue types, duration of expression, and the like. The choice of components and their molar ratios can dramatically affect the encapsulation efficiency of nucleic acids, the stability of LNPs, cellular uptake, endosomal escape, and release profile of the payload. Zhang et al., 2021; Wang et al., 2015.

[0144] More importantly, screening for in vivo targets represents an even greater challenge due to throughput limitations. In developing this multi-step compositional screening process, we successfully identified top performing LNP formulations from a designed library of 1,080 formulations for liver-targeted transfection through iv administration. In this designed screening process, both in vitro and in vivo steps are considered, which allows for rapid identification of effective formulations through a programmatic approach and greatly reduces animal use. This process can be easily transferred to the development of delivery systems for other nucleic acid payloads, including but not limited to mRNA, siRNA, and miRNA, as well as alternative routes of administration, such as oral, subcutaneous, and inhalation delivery.

[0145] Previous reports on LNP-enabled gene delivery systems claim that the composition of LNPs influences tissue targeting and transfection. Cheng et al., 2020; Zhang et al., 2021; Wang et al., 2015; Cheng and Lee, 2016. The subject matter of the present disclosure reveals that the preferentially high transfection efficiency in the liver mediated by selected LNP formulations is not directly related to in vivo biodistribution or cellular uptake efficiency. Rather, intracellular trafficking steps, including endolysosomal escape and pDNA release, play a much more important role. For LNPs with similar properties and distribution among different organs and tissues, it is entirely possible to confer different tissue-specific transfection outcomes and / or result in different transfection levels among different cell types. This outcome is most clearly seen in the direct comparison between the top performing formulations (DI-3, DI-8, FIII-7, and FIII-8) and selected formulations with similar properties but less potent properties (DI-6 and FIII-1). These findings further emphasize the need for a reasoned, multi-step screening approach when evaluating LNP formulations for each specific target organ and genetic medicine application.

[0146] For therapeutic gene delivery, pDNA as a therapeutic payload offers unique benefits compared to mRNA cargo, including more sustained transgene expression, higher stability, and lower production costs. The results also showed that optimized pDNA LNPs resulted in sustained transgene expression for up to 4-5 days, as opposed to a rapid decline over 2 days. Activation of innate immunity against pDNA has been reported to induce gene silencing and inflammatory responses. Previously, CpG depletion in the pDNA sequence has been explored to address this issue. Here, a novel approach is demonstrated by co-administration of anti-inflammatory siRNAs with pDNA in the same LNP formulation, which can effectively extend transgene expression independent of pDNA sequence modification. Inclusion of anti-inflammatory siRNAs reduced immune cell recruitment and the number of apoptotic cells following treatment with LNPs. Even when STAT and NF-κβ levels were only moderately reduced, this approach results in a significant improvement in the overall level and duration of transgene expression in the liver. This strategy does not require sequence modifications or complex delivery vehicles and can be easily adapted to other delivery systems and applications.

[0147] 1.5 Summary

[0148] Overall, the subject matter of the present disclosure provides a multi-step composition screening platform that allows the best performing pDNA LNPs for liver-specific transgene expression to be rapidly and programmatically identified from an LNP library of over 1,000 formulations. This platform combines in vitro and in vivo screening strategies; it can be extended to other carrier systems and potentially for various routes of administration. In addition, it has been revealed that the preferential transduction of selected LNPs in the liver over other organs / tissues is not directly related to the target in vivo distribution or intracellular uptake efficiency of LNPs; rather, intracellular trafficking events, including lysosomal escape, DNA release, and the like, play a much more important role. Although it can be assumed that LNPs with similar physical properties will be distributed in a similar manner among different organs; they show tissue-specific differences in transfection to all of the different cell types due to differences in intracellular cellular trafficking efficiency in a composition-dependent manner. Finally, an innovative strategy of co-delivering anti-inflammatory siRNA and pDNA has been developed to further extend the expression of pDNA therapy. This LNP-based co-delivery strategy further highlights the unique benefits of enhanced transgene expression profiles using pDNA delivery and offers new opportunities for pDNA-based genetic medicine applications.

[0149] 1.6 Materials and Methods

[0150] 1.6.1 Materials

[0151] DLin-MC3-DMA was purchased from MedKoo Biosciences. DSPC, DOPE, DOTAP, DDAB, 18PG (sodium salt) and 14PA (sodium salt) were purchased from Avanti Polar Lipids. Cholesterol was purchased from Sigma. DMG-PEG (MW2000) (DMG-PEG2000) was purchased from NOF America Corporation. Reporter lysis buffer and luciferin assay solution were purchased from Promega. All pDNA was purchased from Aldevron. D-Luciferin (sodium salt) was purchased from Gold Biotechnology.

[0152] 1.6.2 Cell culture and high-throughput screening for transfection studies

[0153] For monolayer culture studies, HepG2 cells (American Type Culture Collection, USA) were seeded in 96-well plates at a cell density of 10,000 per well one day before transfection. The prepared particles were then added at 1 μg of pDNA mL -1 Pipette into EMEM medium at a final particle concentration of 25 μg pDNA mL -1 8 μL of the particle suspension was pipetted into 200 μL of culture medium in the wells. Incubation continued for 24 h to allow transgene expression. When characterizing luciferase as a reporter, cells were lysed with reporter lysis buffer (Promega) using two freeze-thaw cycles, and lysates were characterized by a luminometer against a standard curve generated using luciferase samples (Promega) with the addition of luciferin assay solution (Promega).

[0154] 1.6.3 Synthesis and characterization of LNPs

[0155] The organic phase was prepared by dissolving a mixture of helper lipids (DOTAP, DDAB, DOPE, DSPC, 14PA, 18PG) (Avanti), cholesterol (Sigma-Aldrich), DMG-PEG2000 (Avanti) and Dlin-MC3 DMA in ethanol at a predefined molar ratio. The aqueous phase was prepared with 25 mM magnesium acetate buffer (pH 4.0, Fisher) along with Luc pDNA (Firefly mLuc, Translate), mCherry pDNA, Cre pDNA or Cy5-labeled pDNA. All pDNAs were stored at -20°C and thawed on ice before use. For high-throughput screening, LNPs were prepared in 96-well plates or 1.5 mL microcentrifuge tubes by adding the ethanol phase directly to the aqueous phase. For in vitro screening, LNPs were directly incubated with cells without dialysis. For in vivo batch analysis screening, LNPs from each cluster were mixed and dialyzed against DI water before injection into mice. For larger scale LNP production, the ethanol and aqueous phases were mixed in a 3:1 ratio in the FNC device using a syringe pump as previously described. The resulting LNPs were dialyzed against DI water in 100,000 MWCO cassettes (Fisher) for 24 h at 4°C and stored at 4°C before injection. For in vitro cellular uptake studies, LNPs were incubated with serum at a volume ratio of 2:1 (LNP / serum) for 30 min at 37°C. The size, polydispersity index and zeta potential of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Diameters are reported as intensity averages.

[0156] 1.6.4 Animals and primary cells

[0157] All animal procedures were performed in accordance with ethical regulations and approval from the Johns Hopkins Institutional Animal Care and Use Committee. Female BALB / c mice (6-8 weeks) were obtained from the Jackson Laboratory, and Ai9 mice were housed at the Johns Hopkins Animal Facilities and randomly grouped. LNPs were injected i.v. via the lateral tail vein of mice at a pre-determined dose per mouse, or intrahepatically via a small incision below the sternum. LNP suspensions were filtered at 200 μg mL through Amicon Ultra-2 centrifugal filter units with a MWCO of 100 kDa. -1 pDNA was concentrated to 30 mg mL -1 Mice were intraperitoneally injected with 100 μL of D-luciferin solution and anesthetized using 1.5% isoflurane in oxygen in a ventilated anesthesia chamber and imaged using an in vivo imaging system (IVIS, PerkinElmer) 5 min after injection. Luminescence was quantified using Living Image software (PerkinElmer). For experiments in Ai9 mice, Cre pDNA LNP formulations were prepared as above and administered by iv injection at a pDNA dose of 25 μg per mouse (n=2). After 3 days, mice were sacrificed and major organs were imaged using IVIS. For in vitro transfection in primary hepatocytes, the Hepatocyte Isolation System (tissue dissociation / cell isolation), BioAssay TM (Bioassay, TM Cells were isolated using a Cat. H2006-02 kit according to the manufacturer's protocol. Hepatocytes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2.

[0158] 1.6.5 Cell isolation and staining for flow cytometry

[0159] To quantify mCherry+ or tdTom+ cells among different cell types in each organ, cell isolation and staining were performed, followed by flow cytometry analysis. For hepatocyte isolation, a two-step collagenase perfusion was performed as described previously. Briefly, mice were anesthetized using isoflurane and then fixed. Perfusion was first performed for 7-10 min using Liver Perfusion Medium (Thermo Fisher), then switched to Liver Digestion Medium (Thermo Fisher) and perfusion was performed for another 7-10 min. Livers were harvested into a plate containing 10 mL of Liver Digestion Medium and dissected to release hepatocytes. The released hepatocytes were then collected and washed with ice-cold Hepatocyte Wash Medium (Thermo Fisher) and centrifuged at 50 × g for 5 min. The supernatant was decanted and the pellet was resuspended in ice-cold Hepatocyte Wash Medium. The cell suspension was passed through a 100 μm filter. The hepatocyte suspension was washed twice with ice-cold hepatocyte washing medium and centrifuged (50 × g) once in PBS for 5 min. Afterwards, the hepatocytes were further strained through a 100 μm filter and centrifuged at 50 × g for 5 min, and the cells were resuspended in 500 μL of staining buffer. The antibodies used here were Brilliant Violet 605 anti-mouse CD45, Cyanine 5 anti-mouse CD326, Alexa Fluor 488 anti-mouse CD31, PerCP-Cyanine 5.5 anti-mouse CD11b (BioLegend), and APC anti-mouse CD11c. Flow data were acquired on an SH800 and analyzed using FlowJo software.

[0160] For the isolation and staining of spleen cells, the removed spleen was finely minced using a sterile blade and diluted with 250 μL of digestion medium (45 U / L). -1 Collagenase I, 25 units μL -1 DNase I and 30 units μL-1 The spleen solution was transferred to a 15 mL tube containing 5-10 mL of digestion medium. The spleen solution was then filtered using a 70 μm filter and washed once with PBS. The cells were pelleted at 300×g for 5 min at 4° C. and resuspended in 2 mL of red blood cell lysis buffer (BioLegend) and incubated on ice for 5 min. After incubation, 4 mL of cell staining buffer (BioLegend) was added and centrifuged again at 300×g for 5 min. The cell pellet was washed three times with staining buffer and stained with antibodies (total volume 100 μL) for 20 min at 4° C. in the dark. The stained cells were washed twice with 1 mL of PBS and then resuspended in 500 μL of PBS for flow cytometry analysis. Antibodies used included Brilliant Violet 605 anti-mouse CD45 (BioLegend), PerCP-Cyanine 5.5 anti-mouse CD11b (BioLegend), APC anti-mouse CD11c, FITC anti-mouse CD3 and PE-Cyanine 7 anti-mouse CD19 (BioLegend).

[0161] For isolation and staining of lung cells, the isolated lungs were minced using a sterile blade and then diluted with 10 mL of 2x digestion medium (90 units μL -1 Collagenase I, 50 units μL -1 DNase I, and 60 units µL -1 The tissue was transferred to a 15 mL tube containing 100 mL of hyaluronidase (100 mL) and incubated at 37°C for 1 hour with shaking. After incubation, any remaining lung tissue was homogenized. The next step was the same as in the spleen protocol above. The antibody used here was the same as that for the hepatocytes.

[0162] 1.6.6 Quantitative assessment of endosomal escape by cellomics analysis

[0163] B16F10 cells expressing GFP-binding galectin-8 (GFP-Gal8) were obtained by transfection with plasmids encoding Piggybac transferase (Hera BioLabs) and Piggybac transposon-GFP-Gal8 (Addgene) and poly(β-amino ester) (PBAE) carrier (Karlsson et al., 2020) and then sorted twice on an SH800 cell sorter (Sony). Cells were cultured in DMEM supplemented with 10% FBS at 100,000 cells per well. Particles were dosed after 24 h as described above. After incubation for the predefined times, cells were washed three times with PBS, fixed with 4% paraformaldehyde (PFA) solution, stained with Hoechst 33342, and then washed three times with PBS. Plates were analyzed by the CellInsight CX7 High-content Analysis (HCA) platform (Thermo Fisher Scientific). Analysis was performed according to a method previously reported in Hu et al., 2019.

[0164] Briefly, imaging was performed on a 501.2 x 501.2 μm 2 1104 x 1104 pixels per field of view, corresponding to an area of 2 The experiments were performed at 20x magnification with a resolution of 10x. A total of 30 fields were analyzed within each well of the plate; and well-averaged results were generated by averaging all cells from all fields in each well.

[0165] 1.6.7 Statistical analysis

[0166] Two-tailed Student's t-test or one-way analysis of variance (ANOVA) was performed when comparing two or more groups, respectively. Statistical analysis was performed using Microsoft Excel and Prism 7.0 (GraphPad). Differences were considered significant when P<0.05 (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0167] [Table 1]

[0168] [Table 2]

[0169] [Table 3]

[0170] [Table 4]

[0171] [Table 5]

[0172] [Table 6]

[0173] Example 5

[0174] Optimizing the composition of mRNA-lipid nanoparticles to enhance Th1 / Th2 immune activation profile and anti-cancer immunity

[0175] 5.1 Overview

[0176] Lipid nanoparticles (LNPs) have been successfully engineered as immune-stimulating delivery platforms for antigen-encoding mRNA for cancer immunotherapy. Major efforts have focused on engineering LNPs to promote transfection and maturation of antigen-presenting cells and modulate TLR-mediated adjuvant activity to enhance CD8+ T cell responses and antitumor effects. Here, we employed a multi-step screening approach to optimize the types and component ratios of helper lipids in an mRNA LNP library to efficiently deliver antigen-encoding mRNA and modulate the immune activation profile, i.e., the balance between Th1 and Th2 responses. First, by screening for dendritic cell maturation and antigen presentation in vitro, followed by evaluating the in vivo suppression of immune activation and tumor growth, we identified LNP formulations with potent antitumor effects, especially in combination with immune checkpoint blockade therapy. Furthermore, our results revealed that LNPs exhibiting strong dual immunostimulatory activity in both type 1 T helper (Th1) and Th2 responses produced the most potent antitumor effects by inducing a coordinated attack by dual T and NK cells. These findings highlight the important role played by LNP composition in addition to payload in tailoring immune responses and provide new opportunities to optimize RNA-based cancer and infectious disease treatments.

[0177] 5.2 Background

[0178] In response to the sudden outbreak of the coronavirus pandemic, two mRNA vaccines against SARS-CoV-2, Spikevax (R) (Spikebacks) (Moderna) and Comirnaty (R)The successful deployment of Comirnaty (BioNTech / Pfizer) clearly validated the safety and efficacy of lipid nanoparticle (LNP) delivery platform in mRNA vaccine modality (Huang et al., 2021; Huang et al., 2022; Wouters et al., 2021). LNPs, as a potent vehicle to mediate the expression of mRNA encoding antigen of interest, have been shown to elicit strong antibody and memory B cell responses (Sahin et al., 2021). In this case, the antigen is primarily expressed by cells at the injection site and then internalized and processed by antigen presenting cells (APCs) such as dendritic cells (DCs) to generate strong antibody and T helper cell responses. Sahin et al., 2021; Wang et al., 2021; Lederer et al., 2020; Alameh et al., 2021; Turner et al., 2021; Hou et al., 2021; Sahin et al., 2014, Pardi et al., 2018; Kowalski et al., 2019.

[0179] The strong efficacy was attributed to the adjuvant activity of LNPs, especially their ability to induce germinal center formation and follicular helper T (Tfh) cell responses. Wang et al., 2021; Lederer et al., 2020; Alameh et al., 2021. In addition, the mRNA LNP vaccines showed a significant increase in CD4 + Not only Th1 cells but also IFNγ + or IL2 + CD8 + Evidence was reported that T cells were also generated. Sahin et al., 2021. These reports suggest that LNPs optimized for COVID-19 mRNA vaccines can stimulate CD4 + Th1 and CD8 +Supporting the concept that cell-mediated immunity may also be generated, Laczko et al., 2020, which may contribute to the Th2 response required for a strong humoral response, generating high levels of neutralizing antibodies (Painter et al., 2021; Lozano-Rodriguez et al., 2022; Reichmuth et al., 2016; Hassett et al., 2021; Pilkington et al., 2021; Miao et al., 2021).

[0180] Although Th2 and Th17 responses are essential for generating strong humoral responses and eradicating extracellular pathogens (Sankaradoss et al., 2022; Bretscher, 2014; Bretscher, 2019; Del Prete, 1998), a growing number of reports have demonstrated that complementing a strong Th2 response with Th1-mediated cellular immunity can not only eliminate SARS-CoV-2-infected cells but also enhance humoral responses (Duarte et al., 2021; Kyriakidis et al., 2021; Zhang et al., 2021; Mulligan et al., 2020; Polack et al., 2020).

[0181] LNPs have previously been tested for delivery of mRNA vaccines to treat cancer and prevent infections, including Zika virus (Alameh et al., 2020), influenza (Lindgren et al., 2017), flavivirus (VanBlargan et al., 2018), HIV (Pardi et al., 2019), and others of the same type.

[0182] It has become clear that a tailored immune activation profile is required to induce a strong antigen-specific immune response. When the delivered antigen is expressed in DCs or other APCs, it generates peptide epitopes that are loaded and displayed in the context of MHC class I molecules, and are then activated by CD8+ T cells are activated. Hou et al., 2021; Sahin et al., 2014; Pardi et al., 2018; Kowalski et al., 2019; Sahin et al., 2020; Miao et al., 2019; Oberhardt et al., 2021; Oberli et al., 2017; Karmacharya et al., 2022; Guevara et al., 2020.

[0183] Strong cytotoxic CD8 + T cell responses and type 1 helper T cell (Th1) immune responses are essential for the design of effective tumor vaccines, leading to the elimination of intracellular pathogens and cancer cells. Miao et al., 2019. Studies have shown that LNP preparations promote the transfection and maturation of DCs (dendritic cells), macrophages and neutrophils, and modulate TLR-mediated adjuvant activity, for the purpose of enhancing CD8+ T cell responses and antitumor effects. However, the composition, therapeutic efficacy, and immune activation profile of mRNA-LNPs, especially the correlation in the balance between Th1 and Th2 responses, and the cooperation with NK cell-mediated cell killing, remain elusive.

[0184] LNP systems offer distinct benefits in terms of structural diversity provided by the diverse lipid composition and broad transfection capabilities across a wide range of cell populations. Cullis and Hope, 2017; Zhang et al., 2021; Lokugamage et al., 2021; Wang et al., 2015; Cheng et al., 2020; Dilliard et al., 2021; Patel et al., 2019; Patel et al., 2022; Swingle et al., 2022; Zhu et al., 2022. Most of the LNP-based nucleic acid delivery platforms commercially available or investigated in clinical studies consist of four or five lipid components: a helper phospholipid (e.g., 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)), an ionic lipid, cholesterol, a PEGylated lipid, and a selective organ-targeting lipid. Cheng et al., 2020; Li et al., 2015; Cheng and Lee, 2016; Kulkarni et al., 2017.

[0185] Recent studies have reported that the choice of lipid components and the relative ratio of lipid components in the formulation significantly affect in vivo transfection efficiency and tissue-specific delivery. Cheng et al., 2020; Dilliard et al., 2021; Zhu et al., 2022; Dobrowolski et al., 2022; Xue et al., 2022. Despite recent advances in mRNA LNP-based vaccines, detailed analysis of the impact of helper lipid charge and relative ratio of LNP components on transfection of different cell populations at the administration site is lacking, which may play a key role in determining antigen expression levels in APCs, immune activation profiles, and therapeutic efficacy. Without intending to be bound by any particular theory, it was believed that distinct immune activation profiles could be generated by tailoring the LNP composition to modulate the transfected cell populations.

[0186] Here, we screened 1,080 LNP formulations for transfection efficiency APCs with the goal of inducing robust cellular immunity, and identified a cohort of formulations with the highest transfection efficiency and antigen-presenting capacity in bone marrow-derived dendritic cells (BMDCs). Selected formulations were further tested for transgene expression levels and immune response induction after subcutaneous (sc) or intramuscular (im) injection. We showed that by varying the LNP composition, it is possible to balance immune stimulation between Th1 and Th2 immune responses. We also explore the feasibility of using one LNP formulation to elicit such a dual attack effect in eliciting both strong Th1- and Th2 immune responses, and improved antitumor efficacy compared to formulations with only Th1-biased immune responses. Furthermore, we confirmed the synergistic effect of combining an optimized mRNA LNP vaccine and global immune checkpoint blockade therapy in a melanoma treatment model; and investigated the mechanisms underlying the enhanced immune response and the correlation between cellular transfection activity and local immune activation profile.

[0187] 5.3 Results

[0188] 5.3.1 LNP Screening for mRNA Delivery, Antigen Presentation and Maturation in Dendritic Cells

[0189] To generate a diverse 1,080 LNP library, we used DLin-MC3-DMA as the ionic lipid, DMG-PEG2000 as the PEGylated lipid, and six helper phospholipids previously used in FDA-approved or experimental LNP formulations (Zhu et al., 2022). These were chosen to represent different charges: cationic lipids: 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP) and dimethyldioctadecylammonium (DDAB); zwitterionic lipids: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DSPC; anionic lipids: 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (18PG). Cheng et al., 2020; Dilliard et al., 2021; Patel et al., 2022; Cheng and Lee, 2016; Dobrowolski et al., 2022; Xue et al., 2022; Zhang et al., 2022.

[0190] DLin-MC3-DMA, cholesterol, DMG-PEG2000, and one of six helper lipids were used to generate 1,080 final LNP formulations by varying the following parameters: (1) the combined molar percentages of DLin-MC3-DMA and helper lipid ranging from 20% to 80%, (2) the weight ratio of cholesterol to DMG-PEG2000 ranging from 10 to 500, (3) the weight ratio of DLin-MC3-DMA to helper lipid ranging from 1 to 200, and (4) the molar ratio of charged groups on the ionizable lipid to phosphate groups on the pDNA (N / P ratio) ranging from 4 to 12. The selection of these parameters provided a library of LNP formulations diverse enough to evaluate mRNA delivery (Figure 22a).

[0191] To select LNP formulations with strong APC-specific transgene expression, we first evaluated the mRNA delivery efficiency of the LNP library in DC2.4 cells (immortalized murine dendritic cell line) using firefly luciferase (fLuc) mRNA and measured luciferase protein expression (Figure 22b). The gene expression level varied significantly with the four parameters of the above LNP formulations, including immobilizing helper lipids. We then verified the transfection efficiency of the top 49 LNP formulations containing mCherry mRNA in BMDCs using flow cytometry analysis (Table 7). The results shown in Figure 22c confirmed the high in vitro transfection efficiency of these LNPs. Seven LNP formulations, including C3, C9, C10, D1, D2, D6, and F5 (indicated by red arrows), achieved strong transfection of BMDCs, with over 70% of cells transfected (Table 8).

[0192] An effective mRNA vaccine requires both efficient antigen expression and potent immune cell activation to generate a robust immune response. Therefore, the immunostimulatory effects of seven selected formulations were tested on BMDCs using mRNA (mOVA) encoding ovalbumin, a model antigen protein widely used in vaccine research. After 72 h treatment with mOVA LNPs, compared with PBS, OVA protein, and other formulations, C10, D6, and F5 LNPs resulted in significantly elevated expression of OVA-derived SIINFEKL peptide on MHC-I, indicating successful antigen presentation (approximately 30% SIINFEKL-H-2Kb in C10). + , approximately 21.5% for D6, and approximately 14.0% for F5) (Fig. 22d). In addition, after treatment with these three LNPs, the CD86 + SIINFEKL-H-2Kb + and CD40 + SIINFEKL-H-2Kb + Significantly higher levels of CD40 were observed (Figures 22e-g). Compared to the positive control group treated with lipopolysaccharide (LPS) and SIINFEKL peptide, F5 significantly increased the levels of CD40+ SIINFEKL-H-2Kb + (approximately 5.0%) to comparable levels, and C10 and D6 produced 1.43- and 1.22-fold higher levels, respectively. Along with the enhanced maturation levels of BMDCs, significantly higher secretion levels of the inflammatory cytokines IFN-γ, TNF-α, and IL-6 were also observed in the supernatants of BMDCs after treatment with the three OVA mRNA-loaded LNPs (C10, D6, and F5 LNPs) (Figure 22h).

[0193] We next evaluated the in vivo delivery efficacy of these candidates after subcutaneous or intramuscular injection in mice. To determine whether transfected APCs are present in the draining lymph nodes, we delivered Cre recombinase mRNA (mCre) LNPs into genetically engineered tdTomato (tdTom) reporter mice (Ai9 mice) that contain a LoxP-flanked STOP cassette that prevents expression of tdTom protein. This mouse model is able to detect transfected cells as a result of Cre recombinase expression (Figure 23a), because the expressed recombinase edits the STOP cassette to allow expression of fluorescent tdTom. Our results show that all three LNP candidates (C10, D6, and F5) were able to express tdTom in the draining lymph nodes. + Our results indicated that the nanovaccine produced significant levels of lymphocytes and dendritic cells (DCs) (Fig. 23b, c). Consistent with many reports showing that nanovaccines can efficiently enter and exit draining lymph nodes after subcutaneous administration, our results indicated that a higher percentage of transfected cells was detected in the draining lymph nodes after subcutaneous injection compared to intramuscular injection (Fig. 23b, c). Based on this result, the subcutaneous administration route was chosen for further studies.

[0194] Next, we examined the antigen presentation and maturation levels of APCs in the draining lymph nodes after subcutaneous injection of LNPs (Figure 23d). After three doses of mRNA LNPs, all three preparations significantly increased the levels of SIINFEKL-H-2K in the draining lymph nodes.b+ The DC levels of SIINFEKL-H-2K were significantly improved; 2.9-fold, 2.0-fold, and 1.7-fold higher in D6, F5, and C10, respectively, compared with the free OVA protein-treated group. b+ A DCs level of CD86 was observed (Fig. 23e). + SIINFEKL-H-2K b+ The abundance of DCs was also increased by 2.7-fold, 2.5-fold, and 2.8-fold after treatment with D6, F5, and C10, respectively, compared with the free OVA protein-treated group (FIG. 23f).

[0195] Taken together, the above results demonstrated the successful identification of the top 49 preparations from a LNP library of 1,080 preparations based on transfection efficiency in DC2.4 cells. Further evaluation on BMDCs led to the selection of seven leading LNPs, and three of them (C10, D6, and F5 LNPs) showed potent antigen-presenting and immunostimulatory effects both in vivo and in vitro. Thus, these three leading preparations were selected for further in vivo testing.

[0196] 5.3.2 Distinct Immunologic Profiles of Top Performing LNPs

[0197] The vaccination potential of the three lead LNPs was further tested in mice after subcutaneous injection (three doses on days 0, 7 and 14) (Figure 23d). For comparison, we used the Moderna COVID-19 vaccine (Spikevax (R) SM-102 LNP formulations and the classical adjuvant aluminum hydroxide gel (Alhydrogel) used in (R) ) (mixed with OVA protein in a 1:1 ratio) was also included in further experiments. First, the antigen-specific CD8 + T cell responses were evaluated. Spleens of vaccinated mice were harvested on day 21 and homogenized into cell suspensions for ex vivo antigen restimulation. For all three LNP-treated groups, CD8 + IFN-γ +, CD8 + Granzyme B + , and CD8 + TNF-α + Increased frequencies of CD8+, CD9+, and CD10+ cell populations were observed after treatment with C10, D6, and F5, respectively, compared to the groups administered with free OVA protein (Fig. 23g-i). + TNF-α + There was an approximately 2.3-fold, 2.9-fold, and 2.9-fold increase in cell frequency. (R) A limited increase in antigen-specific cytotoxic T cells was observed for both the C10, D6 and F5 LNPs treatment groups. Compared to SM-102 LNPs, CD8 + TNF-α + The cell frequencies were approximately 1.4-fold, 1.8-fold, and 1.8-fold, respectively. + Along with the T cell response, Th1 cells (CD4 + IFN-γ + ) were also observed (Fig. 23j). In particular, there was a 2.0-fold higher level of antigen-specific Th1 cells after vaccination with F5 LNPs, and a 1.5-fold increase with both C10 and D6 formulations. In contrast, there was no significant increase for Alhydrogel and SM-102 LNP-treated groups. Compared to SM-102 LNPs, there was a 1.3-, 1.4-, and 1.8-fold increase in the levels of antigen-specific Th1 cells after treatment with C10, D6, and F5 LNPs, respectively. Enhanced production of inflammatory cytokines, including IFN-γ, TNF-α, and IL-6 after restimulation with antigen, also points to a strong antigen-specific Th1-type response induced by the three major LNP formulations (Fig. 2k).

[0198] For these groups, antigen-specific CD4 + IL-4 +The frequency of Th2 cells and the magnitude of the antibody response generated were further examined. Compared with the free OVA protein-treated group, significantly more Th2 cells (5.8-fold higher, P=0.0003) were observed in the C10-treated group, in contrast to the D6 and F5 groups (1.8-fold and 1.1-fold, respectively, P>0.5) (Fig. 23l). In addition, as shown in Fig. 2m, C10 induced significantly higher OVA-specific IgG titers, including both IgG1 and IgG2a subclass titers, indicating a strong humoral response. However, the antibody response generated by D6 was limited, and it was not detectable in F5. Alhydrogel (R) Strong OVA-specific IgG titers were observed 3 weeks post-vaccination for both the SM-102 LNP and SM-102 LNP groups.

[0199] The above results show that after vaccination, all three selected LNPs were significantly more effective than Alhydrogel (R) These results indicate that the C10 LNPs successfully induced stronger antigen-specific Th1-type immune responses than the SM-102 LNPs and SM-102 LNPs. However, for Th2-type responses, the three main LNP formulations showed distinct profiles. C10 LNPs elicited both strong Th1 and Th2 responses. While D6 and F5 generated strong Th1 responses, they generated only moderate or undetectable levels of Th2 responses.

[0200] 5.3.3 Differences in antitumor efficacy among top performing LNPs

[0201] Given the strong antigen-specific immune responses induced by the top mRNA LNPs, we investigated the efficacy of the three LNP formulations as cancer vaccines in multiple prophylactic and therapeutic tumor models. C57BL / 6 mice were injected with 10 μg of free OVA protein, Alhydrogel on days 0, 7, and 14. (R) On day 21, animals were immunized with LNPs containing 1 × 10 6All three LNP formulations, together with SM-102 LNPs, showed increased overall survival time with free OVA protein and Alhydrogel. (R) The median survival times were 40, 32, 30, and 32 days for the C10, D6, F5, and SM-102 LNP groups, respectively, compared with 15 days for the free OVA protein group and 15 days for the Alhydrogel group. (R) In addition, compared to D6 and F5, which generated only a strong Th1 response, or SM-102 LNPs, which generated only a strong Th2 response, C10 LNPs, which induced both Th1 and Th2 responses, provided significantly improved protection, with around 40% of mice remaining tumor-free beyond 60 days.

[0202] We next evaluated the therapeutic efficacy of C10 LNPs as a therapeutic vaccine in the Bl 6F10 tumor model using the model OVA antigen, as well as two other clinically relevant antigens, tyrosinase-related protein 2 (Trp2) and glycoprotein 100 (Gp100). First, C57BL / 6 mice were inoculated with 3 × 10 5 100μg of B16F10-OVA cells were subcutaneously inoculated into the mice. On days 4, 11, and 18, the mice were vaccinated with C10 LNPs containing 10μg of mOVA (Fig. 24e). As demonstrated in Fig. 24f-h, C10 LNPs showed a significant tumor suppressive effect in this treatment model, with a median survival time of 26 days compared to 16 days for the negative control group. When C10 was administered in combination with an immune checkpoint inhibitor (anti-CTLA-4 monoclonal antibody 100μg, intraperitoneally administered on days 6, 13, 20, and 27), a synergistic effect was observed, with an extended median survival time of 33.5 days. In contrast, no significant tumor suppressive effect was observed in the group treated with α-CTLA-4 antibody alone, compared to the PBS control.

[0203] Next, C10 LNPs were tested in the same mouse model using the clinically relevant tumor antigens Trp2 and Gp100 rather than the model antigen OVA (Figure 24i). C57BL / 6 mice were injected with 3x10 5 Mice were inoculated subcutaneously in the right hind flank with B16F10 cells from mice. On days 4, 11, and 18, mice were vaccinated with 10 μg of C10 LNPs containing mRNA encoding either Trp2 or Gp100. A strong antitumor effect was also observed using these two antigens, with C10-mTrp2 LNPs and C10-mGp100 LNPs showing significantly extended median survival of 23 and 23.5 days, respectively (Figure 24j-l). However, no significant improvement was observed when these C10 formulations were combined with α-CTLA-4 antibody treatment (Figure 24j-l).

[0204] 5.3.4 Long-term protection correlated with dual T-cell and NK-cell attack

[0205] To further understand the mechanism of efficacy of the mRNA LNP vaccine, we performed cell depletion experiments with C10 and F5 LNPs that induced distinct immunological profiles in the B16F10-OVA melanoma model (Figure 25a). As shown in Figures 25b-25g, CD3 + T cells, NK1.1 + NK cells, or CD20 + Depletion of B cells significantly reduced the survival benefit conferred by C10-mOVA LNPs. In contrast, for F5-mOVA LNPs, the antitumor effect was abolished only when T cells were depleted. Depletion of NK cells or CD20+ B cells did not significantly alter the tumor suppressor effect induced by F5-mOVA LNPs.

[0206] Tumors from mice immunized with C10 LNPs 22 days after tumor inoculation were statistically enriched for various effector lymphocyte populations, with 6.9-fold enrichment for NK cells, 11.2-fold enrichment for T cells, and 11.2-fold enrichment for CD8 cells compared to the PBS control group. +T cells were enriched 7.4-fold (Figure 25h). In addition, C10-mOVA LNPs enhanced the expression of CD8 versus regulatory T cells (T reg ) ratio was significantly increased: 7.8-fold and 3.7-fold higher than those of the PBS group and F5-mOVA LNPs, respectively (Figure 25i). Furthermore, as shown in Figure 25j, higher numbers of NK cells and T cells were observed in tumors from mice treated with C10-mOVA LNPs, whereas no such enrichment effect was detected in mice treated with F5-mOVA LNPs.

[0207] 5.3.5 Potential pathways to induce Thl-only or Thl plus Th2 immune responses

[0208] As shown in Figure 26a, when antigens are translated in the cytoplasm of DCs and the processed peptides are loaded onto MHC-I molecules, a strong cytotoxic T cell response can be initiated along with a Th1 immune response. On the other hand, a Th2 response requires that antigens are expressed and released by non-APCs and then internalized and processed by DCs, macrophages, or B cells, where they are presented in the context of MHC-II. In some situations, antigens from the extracellular environment may be presented on MHC class I molecules via the cross-presentation pathway, but the composition of mRNA LNPs has limited influence on this process. To explore the mechanism for the biased Th1 versus Th2 response generated by GFP mRNA containing LNPs with different compositions, we investigated the local transfection process after subcutaneous injection. At 24 hours after injection, we quantified the GFP expression levels in various cell types at the injection site using flow cytometry (Figure 26b-Figure 26c). After a single injection of D6 or F5 LNPs, around 45% of the transfected cells in the local tissue were immune cells, which was 2-fold higher than that of C10 LNPs. The ratio of immune to non-immune cells among GFP-expressing cells was 4.5 in the C10 group, which was 2.9- and 3.6-fold higher than that of the D6 and F5 groups, respectively. These results indicated significant differences between LNPs with different compositions in terms of transfected cell types.

[0209] To further explore the difference in transfection efficiency in non-immune versus immune cells, we co-cultured C2C12 cells, a mouse myoblast cell line, and BMDCs (1:1) and quantified in vitro delivery to the two cell types using flow cytometry to detect mCherry expression, which revealed that the ratio of C2C12 cells to BMDCs among transfected cells reached as high as 33.41 with C10 LNPs, 22.21 with D6, and only 3.94 with F5 (Figure 26d). When LNPs containing ILuc mRNA were introduced into C2C12 cells alone, the average fLuc expression level in C2C12 cells transfected with C10 was 25- and 289-fold higher than those with D6 and F5 LNPs, respectively (Figure 26e). However, when transfecting pure BMDC cultures with LNPs containing mCherry mRNA, similar transfection efficiencies were observed for C10, D6, and F5 (Figure 26f). Furthermore, we investigated cellular uptake along with transfection efficiency using LNPs containing 50% Cy5-labeled mGFP and 50% unlabeled mGFP. Although nearly 100% Cy5+ cells (i.e., cellular uptake) were observed in all three groups, approximately 27.7% and 8.9% of C2C12 cells were transfected by F5 and D6, respectively, in contrast to 98.1% of cells transfected by C10, which may be caused by differences in endosomal escape capacity among the three LNP preparations in C2C12 myoblasts (Figure 26g-h). When using Lysotracker to visualize lysosomes in C2C12 cells, a lack of colocalization in fluorescence between LNPs (red) and lysosomes (green) was observed for C10 LNPs, suggesting successful lysosomal escape and cytoplasmic delivery (Figure 26i).

[0210] 5.4 Discussion

[0211] The use of LNPs as non-viral gene carriers has progressed rapidly over the past few years, as evidenced by the approval of various LNP-based COVID-19 vaccines and one siRNA therapy (Pardi et al., 2018; Akinc et al., 2019). The safety following repeated LNP dosing provides a strong impetus to extend the utility of LNPs to therapeutic vaccines and other gene delivery applications (Huang et al., 2021; Sahin et al., 2021; Mulligan et al., 2020). Previous reports on LNP-mediated gene delivery have revealed that both the choice of lipids and their molar ratios can dramatically affect the encapsulation efficiency of nucleic acid payloads, transfection efficiency, and cell / tissue targeting profile (Lokugamage et al., 2021; Cheng et al., 2020; Zhu et al., 2022).

[0212] In this study, we screened LNP compositions to investigate the role of the carrier itself in polarizing therapeutic immune responses. We identified three top-performing LNPs from a library of 1,080 candidates based on their transfection efficiency in BMDCs. These three LNPs induced equally strong antigen-specific Th1 responses after three doses by subcutaneous injection, but Th2 responses differed significantly. All three formulations showed significant levels of efficacy in tumor suppression and significantly extended survival in a preventative model of OVA-expressing melanoma in C57BL / 6 mice. However, the best candidate, C10 LNPs, showed the strongest efficacy in slowing tumor growth and extending survival when tested in a therapeutic melanoma model using mRNA encoding OVA, Trp2, or Gp100 antigens.

[0213] Previous reports on cancer vaccine delivery systems demonstrated that a strong Th1 immune response is essential for antitumor efficacy. Badrinath et al., 2022. Using the optimized LNP-delivered mRNA vaccine in this study, we revealed that incorporating a strong Th2 response into a strong Th1 response further improved tumor suppression efficiency by comparing two representative LNP formulations that induce Th1-only or Th1 plus Th2 immune responses. The results confirmed that the coordinated immune response by various cell populations, including T cells, NK cells, and B cells generated by C10-mRNA LNPs, provided even more effective and comprehensive protection against tumor attack, and that the diverse effector cell populations involved in both Th1 and Th2 responses collectively contributed to long-term protection. Although T cell-mediated immunity significantly suppresses tumor growth, many tumors effectively evade the immune system and progress under immune pressure through diverse mechanisms, including loss of MHC class I and the expression and development of an immunosuppressive tumor microenvironment. Our data suggest that if Th2 immune responses are successfully induced, long-term antitumor protection can be achieved by utilizing innate immune cells, such as NK cells, that provide antitumor cytotoxicity activated by antibody-linked target cells. Our data demonstrated that the coordinated action of NK cells and B cells plays an important role in long-term defense against tumors.

[0214] Previous reports on LNP-enabled gene delivery systems claim that the composition of LNPs affects tissue targeting and transfection. Lokugamage et al., 2021; Cheng et al., 2020; Dilliard et al., 2021; Li et al., 2015. We have demonstrated that we can alter the transgene levels delivered by LNP-mediated mRNA vaccines in different cell types in vivo by tailoring the composition of LNP formulations. Furthermore, bias in cell type-specific gene expression was shown to alter the immune activation profile of these formulations. LNPs with strong transfection efficiency in APCs can generate strong cytotoxic T cell and Th1 responses. By varying the composition, LNPs can also show strong transfection efficiency in non-APC cell types such as myoblasts, thereby aiding Th2 responses. These findings further highlight the need for rational screening approaches when evaluating LNP formulations in antigen-specific therapeutic vaccines and other gene medicine applications. This study demonstrated that modulating Th1- versus Th2-biased responses is feasible by altering the composition of LNPs to alter preferential transfection properties in APC and non-APC cell types. This synthetic harmonization approach creates the opportunity to devise novel mRNA LNP-based immunotherapy strategies that can be intricately tailored to specific disease targets, tissues, and cell types.

[0215] The three selected LNP candidates generated different levels of Th2 responses that correlated with different transfection capacities in non-APCs cells such as myoblasts. Among them, C10 LNPs with zwitterionic helper lipid DOPE showed strong Th1 plus Th2 responses and correlated with their higher transfection activity in both DCs and myoblasts, while F5 LNPs with anionic helper lipid showed strong transfection activity only in DCs, resulting in a Th1-biased response. These results indicated that varying the composition of LNP formulations allows preferential transfection activity across various cell types, as exemplified here by myoblasts versus DCs. The difference in endosomal escape ability of different LNP formulations may be a determining factor for the difference in transfection efficiency. This cell type-preferential transfection strategy may be a potential tool to modulate the balance of antigen-specific immune stimulation between Th1 and Th2 responses.

[0216] Overall, we report a compositional screening platform that enabled us to identify the best performing mRNA LNPs for APC-specific transgene expression that exhibited strong Th1 immune responses against tumor antigens in melanoma mouse models. Among the top LNP candidates, C10 exhibited both strong Th1 and Th2 responses, which further enhanced therapeutic efficacy compared to Th1-biased responses against melanoma antigens. The data indicate that coordinated T cell, NK cell, and B cell responses contribute to enhanced antitumor efficacy. In addition, tuning the composition of the LNP formulation altered the transgene levels delivered by LNP-mediated mRNA vaccines in different cell types in vivo. Thus, this study demonstrated a potential strategy to tailor antigen-specific immune activation profiles generated by adjusting LNP composition, providing a versatile vaccine development platform that can be applied to various diseases and leveraged to expand the utility of mRNA LNP-based immunotherapy.

[0217] 5.5 Materials and Methods

[0218] 5.5.1 Materials

[0219] DLin-MC3-DMA was purchased from MedKoo Biosciences. DSPC, DOPE, DOTAP, DDAB, 18PG, 14PA, and DMG-PEG-2000 were from Avanti Polar Lipids. Cholesterol was purchased from Sigma-Aldrich. B16F10 cells (CRL-6475) were purchased from ATCC (American Type Culture Collection, USA). DC2.4 cells and B16F10-OVA (expressing a model antigen, OVA, with a transmembrane domain) were provided by the laboratory of Prof. Jonathan Schneck. Reporter lysis buffer and luciferin assay solution were purchased from Promega. All mRNAs were purchased from (TriLink BioTechnologies). D-Luciferin was purchased from Gold Biotechnology and was prepared by Alhydrogel (R) was purchased from InvivoGen.

[0220] 5.5.2 Cell culture and high-throughput screening for transfection studies

[0221] For monolayer culture studies, DC2.4 cells were seeded in 96-well plates at a density of 10,000 cells per well 1 day before transfection. LNPs were administered at a final concentration of 1 μg mL -1 The mRNA was pipetted into RPMI medium at 25 μg mL -18 μL of the LNP suspension of mRNA was pipetted into 200-μl culture medium in each well. Transgene expression was analyzed after 24 h incubation. When characterizing luciferase as a reporter, cells were lysed using two freeze-thaw cycles with reporter lysis buffer (Promega), and lysates were characterized by adding luciferin assay solution (Promega) and by luminometer against a standard curve generated using luciferase samples (Promega).

[0222] 5.5.3 Synthesis and characterization of LNPs

[0223] LNPs were synthesized by adding the organic phase containing lipids directly to the aqueous phase containing mRNA in 96-well plates or 1.5 mL microcentrifuge tubes for high-throughput screening. To prepare the organic phase, a mixture of Dlin-MC3 DMA, cholesterol (Sigma-Aldrich), DMG-PEG2000 (Avanti), and helper lipids selected from the group consisting of DOTAP, DDAB, DOPE, DSPC, 14PA, 18PG (Avanti) was dissolved in ethanol. For the preparation of SM-102 LNPs, a mixture of SM-102, DSPC, cholesterol, and PEG-DMG in a molar ratio of 50:10:38.5:1.5 was prepared. To prepare the aqueous phase, the corresponding mRNA (fLuc mRNA, GFP mRNA, mCherry mRNA, Cre mRNA, OVA mRNA, Trp2 mRNA, or Gp100 mRNA) was prepared in 25 mM magnesium acetate buffer (pH 4.0, Fisher). All mRNA samples were stored at -80°C and thawed on ice before use. For in vitro screening, LNPs were incubated with cells without dialysis. For larger scale LNP production, the prepared aqueous and ethanolic phases were mixed in a 3:1 ratio in a flash complexation (FNC) device using a syringe pump, Zhu et al., 2022, and purified by dialysis against DI water at 4°C for 24 hours using a 100 kDa MWCO cassette (Fisher), and stored at 4°C before injection. The size, polydispersity index, and zeta potential of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments). Diameters are reported as intensity averages.

[0224] 5.5.4 Animals and primary cells

[0225] All animal procedures were performed under an animal protocol (protocol number MO21E193) approved by the Johns Hopkins Institutional Animal Care and Use Committee. Male and female C57BL / 6 mice, 6-8 weeks of age, were purchased from Jackson Laboratory. Male Ai9 mice, 6-8 weeks of age, were housed at the Johns Hopkins Animal Facilities and randomly assigned to groups. Mice were fed pelleted chow and water with free access. Pelleted chow usually contained 5% fiber, 20% protein, and 5-10% fat. Mice typically ate 4-5 g pelleted chow (120 g / kg body weight) and drank 3-5 mL water (150 mL / kg body weight) per day. The temperature of the mouse room was maintained at 18-26 °C (64-79 °F) and relative humidity at 30-70%, with a minimum of 10 room air changes per hour. Standard shoebox-style cages with corncob bedding were used to house the mice.

[0226] LNPs were administered via sc (right flank) or im (right quadriceps) injection at a pre-defined dose per mouse. LNP suspensions were filtered through Amicon Ultra-2 centrifugal filter units with a MWCO of 100 kDa and 200 μg mL for subcutaneous injection. -1 , 400 μg mL for intramuscular injection -1 In the Ai9 mouse experiments, Cre mRNA LNP preparations were prepared as described above and administered via subcutaneous or intramuscular injection at a dose of 10 μg of mRNA per mouse. After 7 days, mice were sacrificed and draining lymph nodes were collected for flow cytometry analysis.

[0227] 5.5.5 Antibodies, cell separation and staining for flow cytometry

[0228] Antibodies used in this study were: PE-Cyanine 7 anti-mouse CD40 (BioLegend #124622); PerCP-Cyanine 5.5 anti-mouse CD80 (BioLegend #104722); FITC, APC, Brilliant Violet 750 anti-mouse CD11c (BioLegend #117306, 117310, 117357); Brilliant Violet 421 anti-mouse CD86 (BioLegend #105032); PE anti-mouse SIINFEKL H-2KB (ThermoFisher #12574382); FITC, Brilliant Violet 605, Brilliant Violet 750 anti-mouse CD11c (BioLegend #117306, 117310, 117357); Brilliant Violet 421 anti-mouse CD86 (BioLegend #105032); 421 anti-mouse CD45 (BioLegend #103108, 103140, 103134); APC anti-mouse CD3 (BioLegend #100236); FITC, APC, Brilliant Violet 750 anti-mouse CD8 (BioLegend #100706, 100712, BD Biosciences #747502); PerCP-Cyanine 5.5 anti-mouse CD4 (BioLegend #100540); PE anti-mouse IFN-γ (BioLegend #505808); Brilliant Violet 421 anti-mouse IL-4 (BioLegend #504120); PE-Cyanine 7 anti-mouse TNF-α (BioLegend #506324); and APC anti-mouse granzyme B (BioLegend #396408). All antibodies were diluted at a ratio of 1:100 before use.

[0229] For the isolation, restimulation and staining of splenocytes, the spleen was removed and minced using a sterile blade, and then diluted with 250 μL of digestion medium (45 units μL). -1 Collagenase I, 25 units μL -1 DNase I, and 30 units μL -1The cells were homogenized in 100 mL of hyaluronidase (1000 mL). The suspension was transferred to a 15 mL tube containing 5-10 mL of digestion medium and then filtered through a 70 μm filter and washed once with PBS. The cells were pelleted at 300×g for 5 min at 4° C. and resuspended in 5 mL of red blood cell lysis buffer (BioLegend) and then incubated on ice for 5 min. The cells were then pelleted at 300×g for 5 min at 4° C. and washed twice with PBS. The collected cells were then seeded in 12-well plates using RPMI-1640 medium. Splenocytes were incubated with OVA (InvivoGen Cat. vac-pova) and SIINFEKL peptide (InvivoGen Cat. vac-sin) (10 μg mL -1 OVA and 2 μg mL -1 The cells were restimulated in vitro for 12 hours with SIINFEKL (Biotin). After restimulation, the cells were harvested and centrifuged at 300×g for 5 minutes. The cell pellet was washed three times with staining buffer and stained with antibodies against surface markers (total volume 100 μL) for 30 minutes in the dark at 4° C. The stained cells were washed twice with 1 mL PBS and then fixed and permeabilized using Fixation / Permeabilization Solution Kit (BD Cat#555028). The cells were then stained with anti-IFN-γ antibody or other antibodies against intracellular cytokines. Flow data were acquired on a Sony SH800 (Sony SH800) and analyzed using FlowJo software.

[0230] For lymph node cell isolation and staining, isolated lymph nodes were mechanically digested through a 70 μm nylon cell strainer to prepare a single cell suspension. The cell suspension was washed once with PBS via centrifugation (300×g) for 5 min. Cells were then resuspended in 100 μL of staining buffer and stained with antibodies (total volume 100 μL) for 20 min in the dark at 4° C. Stained cells were washed twice with 1 mL of PBS and resuspended in 300 μl of staining buffer for flow cytometry analysis. Flow data were acquired on an SH800 and analyzed using FlowJo software.

[0231] 5.5.6 ELISpot assay

[0232] Multiscreen filter plates (Millipore-Sigma #S2EM004M99) were coated with a specific antibody for IFN-γ (BD Biosciences #551881) and blocked according to the manufacturer's protocol. Then, 1×10 5 Plate isolated splenocytes at 200 x g / mL per well and SIINFEKL peptide (2 µg mL -1 Cells were stimulated with IFN-γ (SIINFEKL) for 24 hours. All tests were performed in duplicate or triplicate and included assay positive controls as well as cells from reference donors with known reactivity. Spots were visualized with a mouse IFN-γ detection antibody (BD Biosciences#551881) followed by incubation with streptavidin-HRP (BD Biosciences#557630) and AEC Substrate (BD Biosciences#551951). Plates were then sent to the SKCCC Immune Monitoring Core for analysis.

[0233] 5.5.7 Enzyme-linked immunosorbent assay (ELISA)

[0234] For antibody detection, groups of C57BL / 6 mice were immunized with different vaccines on days 0, 7, and 14. On day 21, 100 μL blood samples were taken from the tail vein, and the levels of antigen-specific IgG in serum were measured by ELISA. For ELISA, flat-bottom 96-well plates (Nunc) were precoated with OVA protein at a concentration of 2 μg per well in 100 mM carbonate buffer (pH 9.6) overnight at 4°C, which were then blocked with 10% fetal bovine serum (FBS) in PBS-Tween (PBS-T). Sera obtained from immunized animals were diluted 100-fold in PBS-T (PBS-0.05% Tween), pH 7.4, and then serially diluted 4-fold. Undiluted and diluted serum were added to the wells and incubated at 37°C for 2 hours. For labeling, horseradish peroxidase-conjugated goat anti-mouse IgG (Southern Biotech Associates, #1013-05) was used at a dilution of 1:5,000 in PBS-T-10% FBS. After adding horseradish peroxidase substrate, the optical density was measured at a wavelength of 450 nm in an ELISA plate reader (Bio-Rad). Samples were considered positive if their absorbance was twice or more than that of the negative control.

[0235] For cytokine detection, cell supernatants of BMDCs and splenocytes were harvested and the levels of IFN-γ, TNF-α, and IL-6 were measured by ELISA. Supernatants were diluted 1:5. ELISA was performed using an uncoated ELISA kit (Invitrogen) according to the manufacturer's protocol. Optical density was measured at a wavelength of 450 nm in an ELISA plate reader (Bio-Rad).

[0236] 5.5.8 BMDC Isolation, Activation, and Antigen Presentation Assays

[0237] Mice were sacrificed and transferred to a clean bench. Mice were disinfected with 70% ethanol. The skin and muscle of the legs were carefully removed to separate the femur and tibia. The proximal and distal ends of each bone were cut with scissors. The bones were flushed with full medium (RPMI1640, supplemented with 10% FBS and 1% penicillin / streptomycin). Two to three mL of medium was flushed from each side for each bone. The medium containing the cells was filtered through a 70 μm cell strainer and the filtrate was collected. The cell suspension was centrifuged at 200 × g for 10 min at room temperature and the supernatant was discarded. The cells were resuspended in 10 mL of full medium and the cell concentration was determined. The cell suspension was diluted to 3 × 10 6 cells mL -1 The cells were seeded onto ultra-low attachment surface Petri dishes (100 mm × 15 mm) at 10 mL per dish. -1 Add 2 mL of GM-CSF to the culture medium for a final GM-CSF concentration of 20 ng mL -1 Up to 1000 cells were added to each well. Cells were cultured at 37°C and 5% carbon dioxide. Half of the medium containing GM-CSF was replaced every 2 days. On day 6, non-adherent and loosely adherent immature dendritic cells were harvested. The cell suspension was centrifuged at 200g for 10 min at room temperature, and then the supernatant was discarded. Cells were cultured at 5 × 10 cells per well in 24-well plates. 5 The cells were seeded at 100x the volume of 100x the culture medium.

[0238] BMDCs were cultured at 1 μg mL in various LNP formulations. -1OVA mRNA, or PBS, free OVA (InvivoGen Cat. vac-pova), LPS (Sigma-Aldrich, Cat#L6529), or SIINFEKL peptide (InvivoGen Cat. vac-sin) for 24 hours at 37°C in complete medium with 5% CO2; LPS+SIINFEKL peptide was used as a positive control for dendritic cell activation. After co-incubation, BMDCs were collected, washed with FACS buffer (1% BSA, 10% FBS in PBS), and then stained with fluorescently labeled antibodies against CD45, CD11c, CD40, CD80, CD86, and SIINFEKL / H-2Kb monoclonal antibodies on ice.

[0239] 5.5.9 Immunotherapy and Oncology Experiments

[0240] Inject B16F10-OVA cells (1 × 10 in prevention and depletion studies) into the right flank of 6-8 week-old mice. 6 , and in therapeutic studies, 3 × 10 5 ) or 3 × 10 5 of B16F10 melanoma cells were injected subcutaneously. In therapeutic studies, vaccination was initiated when tumor size was less than 50 mm (day 4 after tumor inoculation). Animals were immunized by subcutaneous injection of different LNP formulations containing 10 μg of OVA mRNA, mTrp2, or mGp100 as described in the text. A total of three doses were administered. For combination immunotherapy, some groups were injected intraperitoneally with 100 μg of a checkpoint inhibitor (anti-CTLA-4 mAb) on days 6, 13, 20, and additionally on day 27 for OVA-expressing melanomas after inoculation. Tumor growth was measured three times a week using digital calipers and calculated as 0.5 × length × width × width. For tumor volumes of 2,000 mm 3 Mice were euthanized when they reached

[0241] 5.5.10 Depletion research

[0242] Immune cell depletion was performed with antibodies against NK1.1 (clone PK136, BioXCell), CD3 (clone 145-2C11, BioXCell) and CD20 (clone MB20-11, BioXCell) at 200 μg ip every 4 days. Dosing of all depleting antibodies was started 3 days before tumor inoculation and continued every 4 days.

[0243] 5.5.11 Statistical analysis

[0244] When comparing two groups or more than two groups, a two-tailed Student's t test or one-way analysis of variance (ANOVA) was performed, respectively. Survival curves were compared using the log-rank Mantel-Cox test, and stacked P values ​​were corrected by the Holm-Sidak method for multiple comparisons with alpha set at 0.05. Statistical analysis was performed using Microsoft Excel and Prism 8.0 (GraphPad). Differences were considered significant if P<0.05 (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0245] [Table 7]

[0246] [Table 8]

[0247] References

[0248] All publications, patent applications, patents, and other references mentioned herein are indicative of the level of skill of those in the art relevant to the subject matter of this disclosure. All publications, patent applications, patents, and other references are incorporated herein by reference to the same extent as if each individual publication, patent application, patent, and other reference was specifically and individually indicated to be incorporated by reference. Although a number of patent applications, patents, and other references are referenced herein, it will be understood that such reference is not an admission that any of these documents constitutes part of the common general knowledge in the art. In the event of a conflict between this specification and the incorporated references, the specification (including any amendments thereto that may be based on the incorporated references) shall control. Unless otherwise indicated, standard meanings of terms are used herein. Standard abbreviations are used herein for various terms.

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Claims

1. 1. A solid nanoparticle comprising a steroid, an ionizable cationic lipid, a helper lipid, a PEGylated lipid, and a nucleic acid payload comprising one or more nucleic acids, the solid nanoparticle comprising: a molar ratio of steroid to PEGylated lipid of between about 10 and about 900; a molar ratio of ionizable cationic lipid to helper lipid of between about 1 and about 200; a combined percentage of ionic lipids and helper lipids between about 20% and about 80%; and N to P ratio between about 2 and about 14 The nanoparticles include

2. 2. The solid nanoparticle of claim 1, wherein the steroid comprises a sterol.

3. 3. The solid nanoparticle of claim 2, wherein the sterol comprises cholesterol.

4. The solid nanoparticle of claim 1, wherein the ionizable cationic lipid comprises Dlin-MC3-DMA.

5. 2. The solid nanoparticle of claim 1, wherein the helper lipid is selected from a cationic lipid, a zwitterionic lipid, and an anionic lipid.

6. 6. The solid nanoparticle of claim 5, wherein the cationic lipid is selected from 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and dimethyldioctadecylammonium (DDAB).

7. 6. The solid nanoparticle of claim 5, wherein the zwitterionic lipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

8. 10. The solid nanoparticle of claim 1, wherein the anionic lipid comprises a phospholipid.

9. 9. The solid nanoparticle of claim 8, wherein the phospholipid is selected from 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA) and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

10. 2. The solid nanoparticle of claim 1, wherein the PEGylated lipid comprises dimyristoylglycerol (DMG)-polyethylene glycol (PEG) 2000 (DMG-PEG2000).

11. 2. The solid nanoparticle of claim 1, wherein the one or more nucleic acids are selected from plasmid DNA (pDNA), siRNA, mRNA, and combinations thereof.

12. 2. The solid nanoparticle of claim 1, wherein the one or more nucleic acids include a combination of plasmid DNA (pDNA) and siRNA.

13. 13. The solid nanoparticle of claim 11 or claim 12, wherein the siRNA comprises an anti-inflammatory siRNA.

14. 10. The solid nanoparticle of claim 1, wherein the nanoparticle has a size of less than about 400 nm.

15. 13. A method for delivering one or more nucleic acids to the liver of a subject, comprising administering the solid nanoparticles of claim 1 to a subject in need of treatment.

16. 17. The method of claim 16, wherein the one or more nucleic acids are selected from plasmid DNA (pDNA), siRNA, and combinations thereof.

17. 18. The method of claim 17, wherein the one or more nucleic acids include a combination of plasmid DNA (pDNA) and siRNA.

18. The method of claim 17 or claim 18, wherein the siRNA comprises an anti-inflammatory siRNA.

19. 20. The method of claim 19, wherein the anti-inflammatory siRNA targets a transcription factor selected from signal transducer and activator of transcription (STAT) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ).

20. 20. The method of claim 19, comprising reducing inflammation-induced gene silencing.

21. The method of claim 19, wherein the duration of expression of the pDNA when administered together with the anti-inflammatory siRNA is longer than the duration of expression of the pDNA when administered alone.

22. 23. The method of claim 22, wherein the expression level of the pDNA when administered together with the anti-inflammatory siRNA is substantially similar to the expression level of the pDNA when administered alone.

23. 20. The method of claim 19, comprising reducing liver levels of one or more signal transduction and activator of transcription (STAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κβ), one or more infiltrating inflammatory monocytes, and one or more apoptotic cells.

24. One or more infiltrating inflammatory monocytes express CD45 + Cells and CD11b + The method of claim 24, selected from cells.

25. 17. The method of claim 16, comprising treating one or more diseases or disorders of the liver.

26. 28. The method of claim 27, wherein the one or more diseases or disorders of the liver are selected from genetic liver diseases and inflammatory liver diseases.

27. 29. The method of claim 28, wherein the one or more diseases or disorders of the liver are selected from hemophilia B, hemophilia A, ornithine transcarbamylase (OTC) deficiency, phenylketonuria, acute intermittent porphyria, methylmalonic acidemia, familial hypercholesterolemia, Fabry, MPS type VI, gangliosidosis GM1, Danon disease, GSDla von Gierke, Wilson's disease, Crigler-Najjar, primary hyperoxaluria type 1, and combinations thereof.

28. 17. The method of claim 16, wherein the method for delivering the one or more nucleic acids to the liver of the subject is selected from intravenous (iv) injection, oral, subcutaneous, and inhalation delivery.

29. 13. A method for preparing the solid nanoparticles of claim 1, comprising: (a) preparing an organic phase by dissolving a mixture of steroid, ionizable cationic lipid, helper lipid, and PEGylated lipid in a predetermined molar ratio in a polar, protic solvent; (b) preparing an aqueous phase by dissolving one or more nucleic acids in an aqueous buffer; and (c) combining the organic phase and the aqueous phase to form solid nanoparticles. A method comprising:

30. Polar, protic solvents include C 1 -C 4 32. The method of claim 31 , comprising alcohol.

31. 32. The method of claim 31, wherein the aqueous buffer comprises a magnesium acetate buffer.

32. 32. The method of claim 31, further comprising mixing the organic phase and the aqueous phase in a flash nanocomposite formation (FNC) device.

33. 34. The method of claim 33, further comprising mixing the organic phase and the aqueous phase in a ratio of about 3:

1.

34. 32. The method of claim 31, further comprising dialysing the solid nanoparticles against deionized water.

35. 13. A method for stimulating T helper type 1 (Th1) and / or T helper type 2 (Th2) responses in vivo comprising administering the solid nanoparticles of claim 1.

36. Steroids include cholesterol; Ionizable cationic lipids include DLin-MC3-DMA; PEGylated lipids include DMG-PEG2000; The nucleic acid is mRNA; 37. The method of claim 36, wherein the helper lipid is selected from 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), DSPC, 1,2-dimyristoyl-sn-glycero-3-phosphate (14PA), and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG).

37. The solid nanoparticles are: A combined molar percentage of DLin-MC3-DMA and helper lipid ranging from about 20% to about 80%; a weight ratio of cholesterol to DMG-PEG2000 ranging from about 10 to about 500; a weight ratio of DLin-MC3-DMA to helper lipid ranging from about 1 to about 200; and A molar ratio of charged groups on the ionizable lipid to phosphate groups on the mRNA (N / P ratio) ranging from about 4 to about 12.

38. The method of claim 37, comprising:

38. The solid nanoparticles are: (a) about 30 mol % DOPE, about 30 mol % DLin-MC3-DMA, about 40 mol % cholesterol, about 0.40 mol % DMG-PEG2000, and an N / P ratio of about 4; (b) about 7 mol% DSPC, about 70 mol% DLin-MC3-DMA, about 20 mol% cholesterol, about 0.04 mol% DMG-PEG2000, and an N / P ratio of about 4; or (c) about 5 mol % 18PG, about 55 mol % DLin-MC3-DMA, about 40 mol % cholesterol, about 0.40 mol % DMG-PEG2000, and an N / P ratio of about 12 40. The method of claim 38, comprising:

39. 37. The method of claim 36, which induces an immune response in only Th1, only Th2, or in both Th1 and Th2.

40. 13. A method for treating a disease, disorder, or condition in a subject, comprising administering a therapeutically effective amount of the solid nanoparticles of claim 1 to a subject in need of treatment.

41. 42. The method of claim 41, wherein the disease is selected from cancer or infectious disease.

42. 43. The method of claim 42, wherein the cancer is selected from basal cell carcinoma, bladder cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, esophageal carcinoma, gastric cancer, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma, malignant pleural mesothelioma, Merkel cell carcinoma, metastatic melanoma, non-small cell lung cancer, renal cell carcinoma, small cell lung cancer, squamous cell carcinoma, and urothelial carcinoma.

43. 43. The method of claim 42, wherein the infection comprises a viral infection.

44. 45. The method of claim 44, wherein the viral infection is selected from a coronavirus infection, a Zika virus infection, influenza, a flavivirus infection, and a human immunodeficiency virus (HIV) infection.

45. 37. The method of claim 36, further comprising administering the solid nanoparticles together with one or more immune checkpoint inhibitors.

46. 47. The method of claim 46, wherein the immune checkpoint inhibitor is selected from a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor.

47. 47. The method of claim 46, wherein the one or more immune checkpoint inhibitors are selected from ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab.

48. 13. A vaccine comprising the solid nanoparticles of claim 1.

49. 50. The vaccine of claim 49, wherein the vaccine is a cancer vaccine or an anti-viral vaccine.

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