Lipid nanoparticle formulations capable of migrating to systemic organs following intramuscular administration

Abstract

Lipid nanoparticles comprising an ionizable lipid, a PEGylated lipid, a helper phospholipid, a sterol, and a nucleic acid, the composition of which can be tailored to preferentially localize and transfect cells in specific organs, such as the liver.

Description

LIPID NANOPARTICLE FORMULATIONS CAPABLE OF MIGRATING TO SYSTEMIC ORGANS FOLLOWING INTRAMUSCULAR ADMINISTRATIONCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims benefit of U. S. Provisional Application No. 63 / 750,355 filed January 28. 2025, which is incorporated herein by reference in its entirety.FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under grants EB028239 and AI155313 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] In infectious disease control and cancer therapy, there is increasing interest in achieving localized tissue-specific immunity through the induction of antigen- specific CD8+T cells within the local tissue environment, such as CD8+tissue-resident memory T (TRM) cells. Fernandez-Ruiz et al., 2016; Mueller and Mackay, 2016; Okla et al., 2021; Nizard et al., 2017. These tissue-specific T cells play a critical role in long-term immune surveillance and provide rapid responses upon pathogen re-encounter. Park and Kupper, 2015; Mueller and Mackay, 2016; Dijkgraaf et al., 2019; Ariotti et al.. 2012. Their unique ability to remain localized and maintain a heightened state of readiness makes them particularly effective at controlling pathogens in specific tissues. Schenkel and Masopust, 2014; Jiang et al., 2012; Klonowski et al., 2004; Sallusto et al., 1999. Consequently, the induction of such tissue-specific CD8+T cells has become a key consideration in the development of vaccines targeting tissue-specific infections. Rotrosen and Kupper, 2023.

[0004] Antigen expression within the tissue microenvironment facilitates the recruitment of antigen- specific CD8+T cells to the tissue. Upon antigen recognition and adhesion receptor expression, a subset of these CD8+T cells reside in the tissue in a persistent manner, maturing into the more specialized TRM cell population. Debes et al., 2005; Wakim et al., 2010. This subset of T cells is optimized for localized immune surveillance, exhibiting minimal recirculation, and can effectively control tissue-specific infection or tumor, offering an appealing strategy for long-term localized immunity, particularly for diseases where preventing infection at the organ level is crucial. Fernandez-Ruiz et al., 2016; Holz et al., 2018; Khan et al., 2016; Epstein et al., 2011.143985.601_P18418-02

[0005] Current antigen delivery strategies for achieving local tissue expression, however, often rely on intravenous (i.v.) injection and complex targeting moieties, thus complicating vaccine development. Fernandez-Ruiz et al., 2016; Paunovska et al., 2022; Olsen et al., 2018; Watson et al., (2022); Zhu et al., 2022. Furthermore, non-lymphoid tissue-specific transfection and the subsequent localized immune responses beyond systemic immunity via conventional vaccination methods, such as intramuscular (i.m.) injections, remains underexplored.SUMMARYIn some aspects, the presently disclosed subject matter provides a composition comprising a lipid nanoparticle comprising an ionizable lipid; a PEGylated lipid; a helper phospholipid; a sterol: and a nucleic acid, wherein: (a) a ratio of the ionizable lipid to the helper phospholipid ranging from about 1 to about 200; (b) a ratio of the sterol to the PEGylated lipid ranging from about 10 to about 500; (c) a combined percentage of the ionizable lipid and the helper phospholipid ranging from about 20% to about 80%; (d) a ratio (N / P) of amine groups (N) of the ionizable lipid to phosphate groups (P) of the nucleic acid ranging from about 4 to about 15; and, in some aspects, (e) wherein the lipid nanoparticle is capable of migrating to one or more systemic organs following intramuscular administration at a fraction ranging from about 10% to about 99% of an administered dose, and mediates detectable expression of the nucleic acid in at least one systemic organ.

[0006] In certain aspects, the ionizable lipid comprises ALC-0315.

[0007] In certain aspects, the PEGylated lipid comprises DMG-PEG2000.

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

[0009] In particular aspects, the anionic phospholipid is selected from bis(monooleoylglycero)-phosphate (18BMP) and l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (18PG).

[0010] In particular aspects, the zwitterionic phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In more particular aspects, the zwitterionic phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0011] In particular aspects, the cationic phospholipid is selected from l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyl dioctadecyl ammonium (DDAB).

[0012] In particular aspects, the sterol is cholesterol.243985.601_P18418-02

[0013] In some aspects of the composition: (a) the ionizable lipid has a molar percentage ranging from about 10% to about 60%; (b) the helper phospholipid has a molar percentage ranging from about 0% to about 40%; (c) the sterol has a molar percentage ranging from about 40% to about 90%; (d) the PEGylated lipid has a molar percentage ranging from about 0.1% to about 5%; and (e) an N: P ratio ranging from about 4 to about 15.

[0014] In certain aspects of the composition, the lipid nanoparticle comprises: (a) a molar percentage of about 20% to about 30% ionizable lipid, about 20% to about 30% helper phospholipid, about 40% to about 55% sterol, about 1.5 to about 3.5% PEGylated lipid, and an N: P ratio of between about 8 to about 12; (b) a molar percentage of about 10% to about 20% ionizable lipid, about 1% to about 10% helper phospholipid, about 70% to about 90% sterol, about 0.1% to about 2% PEGylated lipid, and an N: P ratio of between about 8 to about 12; (c) a molar percentage of about 35% to about 55% ionizable lipid, about 5% to about 15% helper phospholipid, about 30% to about 50% sterol, about 0.1% to about 2.5% PEGylated lipid, and an N: P ratio of between about 4 to about 8; (d) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0 % PEGylated lipid, and an N: P ratio of between about 8 to about 10; or (e) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1 to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0% PEGylated lipid, and an N: P ratio of between about 5 to about 9.

[0015] In certain aspects, the lipid nanoparticle comprises: (a) a molar percentage of about 25% ionizable lipid, about 25% helper phospholipid, about 47.5% sterol, about 2.5% PEGylated lipid, and an N: P ratio of about 10; (b) a molar percentage of about 14% ionizable lipid, about 6% helper phospholipid, about 79.2% sterol, about 0.8% PEGylated lipid, and an N: P ratio of about 10; (c) a molar percentage of about 46.3% ionizable lipid, about 9.4% helper phospholipid, 42.7% sterol, about 1.6% PEGylated lipid, and an N: P ratio of about 6; (d) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% sterol, about 0.5% PEGylated lipid, and an N: P ratio of about 10; or (e) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% sterol, about 0.5% PEGylated lipid, and an N: P ratio of about 7.

[0016] In certain aspects, the lipid nanoparticle comprises: (a) a molar percentage of about 30% to about 40% ionizable lipid, about 10% to about 20% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0% PEGylated lipid, and an N: P ratio of between about 5 and343985.601_P18418-029; (b) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 5 and about 9; (c) a molar percentage of about 40% to about 50% ionizable lipid, 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 2 to about 6; (d) a molar percentage of about 40% to about 50% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 8 to about 10; (e) a molar percentage of about 30% to about 40% ionizable lipid, about 10% to about 20% helper phospholipid, about 40% to about 60% sterol, about 0.1 to about 1.0% PEGylated lipid, and an N: P ratio of between about 2 to about 6; or (f) a molar percentage of about 60% to about 80% ionizable lipid, about 5% to about 10% helper phospholipid, about 15% to about 25% sterol, about 0.01% to about 0.1% PEGylated lipid, and an N: P ratio of between about 8 to about 12.

[0017] In certain aspects, the lipid nanoparticle comprises: (a) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% sterol, 0.5% PEGylated lipid, and an N: P ratio of about 7; (b) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 7; (c) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 4; (d) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 10; (e) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% sterol, 0.5% PEGylated lipid, and an N: P ratio of about 4; or (f) a molar percentage of about 72% ionizable lipid, 8% helper phospholipid, 19.96% sterol, 0.04% PEGylated lipid, and an N: P ratio of about 10.

[0018] In certain aspects, the nucleic acid is selected from an antisense oligonucleotide, cDNA, genomic DNA, guide RNA, plasmid DNA (pDNA), vector DNA, mRNA, miRNA, piRNA, shRNA, and siRNA. In particular aspects, the nucleic acid is mRNA.

[0019] In certain aspects, the nucleic acid comprises a polynucleotide encoding a Cas nuclease. In particular aspects, the Cas nuclease comprises Cas9. In certain aspects, the lipid nanoparticle further comprises a small guide RNA or a DNA encoding a small guide RNA.

[0020] In some aspects, the presently disclosed subject matter provides a vaccine comprising a lipid nanoparticle of the composition described herein.443985.601_P18418-02

[0021] In some aspects, the presently disclosed subject matter provides a method for treating a disease, condition, or disorder, the method comprising administering a composition described herein, or a vaccine comprising the composition, to a subject in need of treatment thereof.

[0022] In certain aspects, administration of the composition or the vaccine elicits a specific T cell response and / or cellular immunity. In certain aspects, administration of the composition or the vaccine elicits a tissue-specific immune response at the primary site of infection.

[0023] In certain aspects, the disease, condition, or disorder comprises a disease, condition, or disorder of the lung. In particular aspects, the disease, condition, or disorder of the lung is selected from pneumonia, respiratory syncytial virus (RSV), influenza, tuberculosis, and a coronavirus.

[0024] In certain aspects, the disease, condition, or disorder comprises a cancer. In certain aspects, administering the composition or the vaccine prevents or reduces one or more cancer cells remaining from a post-tumor excision or other surgical cancer treatment from metastasizing to one or more other organs.

[0025] In certain aspects, the disease, condition, or disorder comprises an infectious disease. In particular aspects, the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the liver. In more particular aspects, the disease, condition, or disorder of the liver is selected from malaria, hepatitis B, and hepatitis C.

[0026] In certain aspects, administration of the composition or the vaccine achieves gene expression in selected peripheral tissues and a lower systemic toxicity.

[0027] In certain aspects, the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the lymphatic system. In particular aspects, the disease, condition, or disorder of the lymphatic system comprises human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS). In certain aspects, tissue-resident T cells clear pathogeninvading cells.

[0028] In certain aspects, the method is a prophylactic method of treatment.

[0029] In certain aspects, the administering is via intramuscular (i.m.) injection or subcutaneous (s.c.) injection.

[0030] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Drawings as best described herein below.543985.601_P18418-02BRIEF DESCRIPTION OF THE FIGURES

[0031] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0032] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0033] FIG. la, FIG. lb, FIG. 1c, FIG. Id, FIG. le, FIG. If, FIG. 1g, and FIG. Ih demonstrate induction of tissue- specific T cell responses following i.m. injection of mRNA LNPs. FIG. la, Schematic illustration of LNP trafficking following i.m. injection. FIG. lb, C57BL / 6 mice were given three i.m. injections, three weeks apart, of PBS or CIO LNPs loaded with mOVA (10 pg mOVA per injection). Mice were sacrificed four weeks after the final injection, and their lymphocytes were isolated from spleens and livers for analysis. Splenocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL) and assessed via ELISpot to determine the frequency of IFN-y-producing cells in spot-forming unit (SFU). FIG. 1c, Cellaca MX High-throughput Automated Cell Counter (HACC) was employed to count the lymphocytes within the harvested tissues. Unstimulated splenocytes were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer. The gating strategy is shown in FIG. 10. FIG. Id, Titres of OVA-specific serum IgG on day 70, determined by ELISA. FIG. le, Lymphocytes isolated from the liver were restimulated in vitro and assessed via ELISpot to determine the SFU frequency according to the same method described above. FIG. If-FIG. Ih, Representative flow cytometry plots for determining OVA-specific CD8+T cells in the liver (FIG. If) are shown. Unstimulated lymphocytes isolated from the liver were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 1g), as well as cells for high CD44 and low CD62L (FIG. Ih). Data are from n = 4 biologically independent samples. “CIO” stands for CIO mOVA LNPs. Data were analyzed using an unpaired Ftest. For box plots, the box extends from the 25thto the 75thpercentiles, and the line in the middle of the box is plotted at the median. *P < 0.05, **P < 0.01, ***P < 0.001,0.0001.

[0034] FIG. 2a, FIG. 2b, FIG. 2c, FIG. 2d, FIG. 2e, FIG. 2f, FIG. 2g, FIG. 2h, FIG. 2i, FIG. 2j, FIG. 2k, and FIG. 21 show assessment of mRNA LNP trafficking following i.m. injection and the subsequent immune responses. FIG. 2a, FIG. 2b, Biodistribution at 12 h (FIG. 2a) and 24 h (FIG.643985.601_P18418-022b) post i.m. injection of CIO LNPs (10 pg Cy5-labelled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency at the injection site versus organs, as well as the percentage distribution across individual organs (n = 4). FIG. 2c, FIG. 2d, Total bioluminescence flux in injection site and organs at 24 h (FIG. 2c) and 48 h (FIG. 2d) post i.m. injection of CIO mLuc LNPs (10 pg mLuc per mouse) in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging with IVIS. Data are expressed as the percentage of total flux at the injection site versus organs, as well as the percentage expression across individual organs (n = 4). FIG. 2e, Timeline for the immune activation experiment. C57BL / 6 mice were given three injections (3 i.m. or 2 i.m. + 1 i.v.), three weeks apart, of PBS or CIO LNPs loaded with mOVA (10 pg mOVA per injection). Mice were sacrificed four weeks after the final injection, and their lymphocytes were isolated from spleens and livers for analysis. FIG. 2f-FIG. 2h, The Cellaca MX HACC was employed to count the lymphocytes within the harvested tissues. Representative flow cytometry plots for determining OVA-specific CD8+T cells in the liver (FIG. 2f) are shown. Unstimulated lymphocytes isolated from the liver were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 2g), as well as cells for high CD44 and low CD62L (FIG. 2h). The gating strategy is shown in FIG. 10. FIG. 2i, Lymphocytes isolated from the liver were restimulated in vitro and assessed for the SFU frequency. FIG. 2j, Timeline for OT-I cell migration experiment. CD8+T cells were isolated from OT-I mice and labeled with CellTrace™ Violet (CTV). C57BL / 6 mice were first given one i.m. injection of PBS or CIO LNPs loaded with mOVA (10 pg mOVA per mouse). Six hours post-injection, the same mice were given one i.v. injection (tail vein) of 2.25 x 106CTV-labelled OT-I CD8+T cells. Mice were sacrificed 24 or 48 h post LNP / PBS injection, and their cells were isolated from spleens, livers, and lungs for analysis. FIG.2k, The Cellaca MX HACC was employed to count the lymphocytes within the harvested tissues. Isolated cells were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and CTV in each organ at 48 h post LNP injection. FIG. 21, Biodistribution of CTV-labeled OT-I cells across three major organs in PBS-treated and LNP-treated mice at 48 h. Percentages represent the proportion of OT-I cells from the total number originally injected (2.25xl06). Data are from n = 4 biologically independent samples. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test for FIG. 2g-FIG. 2i, and an unpaired t-test for FIG. 2k. For box plots, the box extends from the 25thto the 75thpercentiles, and the line in the middle of the box is743985.601_P18418-02plotted at the median. For bar plots, data represent mean ± s.e.m. NS: P > 0.05, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.

[0035] FIG. 3a, FIG. 3b, FIG. 3c, FIG. 3d, FIG. 3e, FIG. 3f, FIG. 3g, FIG. 3h, FIG. 3i, FIG. 3j, and FIG. 3k demonstrate systemic trafficking of different LNP formulations following i.m. injection. FIG. 3a, FIG. 3b, Biodistribution at 24 h post i.m. injection of four selected LNPs (10 pg Cy5-labelled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency at the injection site versus major organs (FIG. 3a), as well as the percentage distribution across individual organs (FIG.3b). FIG. 3c, FIG. 3d, Total bioluminescence flux at the injection site and organs 48 h post i.m. injection of four selected LNPs containing 10 pg mLuc per dose in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging using IVIS. Data are expressed as the percentage of total flux at the injection site versus major organs (FIG. 3c), as well as the percentage expression across individual organs (FIG. 3d). FIG. 3e-FIG. 3g, Total flux in photons per second generated by each of the four selected LNPs in the liver (FIG. 3e). lungs (FIG. 3f), and spleen (FIG. 3g) 24 and 48 h post i.m. injection are shown. FIG. 3h, Schematic illustration of Ai9 mouse model used for organspecific transfection validation and timeline of the Ai9 transfection validation experiment. C57BL / 6 mice were given one i.m. injection of selected LNPs loaded 30 pg mCre per mouse. Mice were sacrificed on day 7 post-injection for analysis. FIG. 3i-FIG. 3k, The tdTom+cells were quantified via flow cytometry analysis of the cells isolated from the liver and lungs. Representative flow cytometry plots for tdTom+hepatocytes in the liver of PBT and DL8 groups at 7 days postinjection are shown (FIG. 3i). Percentages of tdTom+cells in the liver (FIG. 3j) and lungs (FIG.3k) are shown. Gating strategies are shown in FIG. 25 and FIG. 26. Data are from n = 4 biologically independent samples. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test for FIG. 3j, FIG. 3k. For bar plots, data represent mean ± s.e.m. NS: P > 0.05, *P <0.05. **P <0.01. ***P <0.001, ****P <0.0001.

[0036] FIG. 4a, FIG. 4b, FIG. 4c, FIG. 4d, FIG. 4e, FIG. 4f, FIG. 4g, FIG. 4h, FIG. 4i, FIG. 4j, FIG. 4k, FIG. 41, FIG. 4m, and FIG. 4n show short-term immune assessments of selected mRNA LNPs following i.m. injection. FIG. 4a, Timeline for the short-term vaccination study. C57BL / 6 mice were given three i.m. injections, one week apart, of PBT, MDN, or DI-8 LNPs containing 10 pg mOVA per injection or PBS. Mice were sacrificed two weeks after the final injection, and their lymphocytes were isolated from the liver, lungs, and spleen for analysis. FIG. 4b-FIG. 4d, The843985.601_P18418-02Cellaca MX HACC was employed to count the isolated lymphocytes within the harvested tissues. Representative flow cytometry plots for determining OVA-specific CD8+T cells in the liver (FIG.4b) are shown. Unstimulated lymphocytes isolated from the liver were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 4c), as well as high CD44, low CD62L, and high CD69 (FIG. 4d). The gating strategy is shown in FIG.30. FIG. 4e-FIG. 4g, Representative flow cytometry plots for determining OVA-specific CD8+T cells in the lungs (FIG. 4e) are shown. Unstimulated lymphocytes isolated from the lungs were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 4f), as well as high CD44, low CD62L, and high CD69 (FIG.4g). FIG. 4h-FIG. 4j, Lymphocytes isolated from the liver, lungs, and spleen were restimulated in vitro with SIINFEKL peptide (2 g / mL SIINFEKL) and assessed via FluoroSpot to determine the SFU frequency in the liver (FIG. 4h), lungs (FIG. 4i), and spleen (FIG. 4j). FIG. 4k, Number of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer in the spleen, assessed via flow cytometry. FIG. 41-FIG. 4n, Titres of OVA-specific IgG. IgGl, and IgG2c antibodies in blood serum on day 28, determined by ELISA. Data represent n = 10 biologically independent samples from three independent experiments (FIG. 4b, FIG. 4e, FIG. 4c, FIG. 4f, FIG. 4k), n = 7 biologically independent samples from two independent experiments (FIG. 4d, FIG. 4g), and n = 4 biologically independent samples from one representative experiment (FIG. 4h-FIG. 4j, FIG. 41-FIG. 4n). Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test for FIG. 4c-FIG. 4d and FIG. 4f- FIG. 4n. For box plots, the box extends from the 25thto the 75thpercentiles, and the line in the middle of the box is plotted at the median. NS: P > 0.05, *P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001.

[0037] FIG. 5a, FIG. 5b, FIG. 5c, FIG. 5d, FIG. 5e, FIG. 5f, FIG. 5g, FIG. 5h, FIG. 5i, FIG. 5j, FIG. 5k, FIG. 5l, FIG. 5m, and FIG. 5n show long-term immune assessments of selected mRNA LNPs following i.m. injection. FIG. 5a. Timeline for the long-term vaccination study. C57BL / 6 mice were given three i.m. injections, one week apart, of PBT, MDN, or DL8 LNPs containing 10 pg mOVA per injection or PBS. Mice were sacrificed 2.5 months after the final injection, and lymphocytes were isolated from the liver, lungs, and spleen for analysis. FIG. 5b-FIG. 5d, The Cellaca MX HACC was employed to count the isolated lymphocytes within the harvested tissues. Representative flow cytometry plots for determining OVA-specific CD8+T cells in the liver (FIG.5b) are shown. Unstimulated lymphocytes isolated from the liver were assessed via flow cytometry943985.601_P18418-02to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 5c), as well as high CD44, low CD62L, and high CD69 (FIG. 5d). The gating strategy is shown in FIG.30. FIG. 5e-FIG. 5g, Representative flow cytometry plots for determining OVA-specific CD8+T cells in the lungs (FIG. 5e) are shown. Unstimulated lymphocytes isolated from the lungs were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 5f). as well as high CD44, low CD62L, and high CD69 (FIG.5g). FIG. 5h- FIG. 5i, Unstimulated lymphocytes isolated from the spleen were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 5h), as well as high CD44, low CD62L, and high CD69 (FIG. 5i). FIG. 5j, Lymphocytes isolated from the spleen were restimulated in vitro with OVA and SIINFEKL peptide (100 pg / mL OVA and 2 pg / mL SIINFEKL) for 12 h and assessed via intracellular cytokine staining and flow cytometry and to determine the percentages of CD3+CD8+TNFoc+cells. FIG. 5k, Lymphocytes isolated from the spleen were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL) and assessed via FluoroSpot to determine the SFU frequency. FIG. 5LFIG. 5n, Titres of OVA-specific IgG, IgGl, and IgG2c antibodies in blood serum on day 90 post- vaccination, determined by ELISA. Data are from representative experiments with n = 4 biologically independent samples. Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test for FIG. 5c-FIG. 5d and FIG. 5f-FIG. 5n. For box plots, the box extends from the 25thto the 75thpercentiles, and the line in the middle of the box is plotted at the median. NS: P > 0.05, *P < 0.05, **P < 0.01, ***P <0.001, ****P <0.0001.

[0038] FIG. 6a, FIG. 6b, FIG. 6c, FIG. 6d, FIG. 6e, FIG. 6f, FIG. 6g, and FIG. 6h demonstrate antitumor efficacy of the mRNA LNP formulations as therapeutic and prophylactic vaccines. FIG.6a-FIG. 6d, Schematic and results of a therapeutic vaccination model for B16-fLuc-OVA in C57BL / 6 mice. Mice were inoculated via intrahepatic injection with B 16-fLuc-OVA cells and then given three i.m. injections, 2 days apart, of PBT, MDN, or DI-8 LNPs containing 10 pg mOVA per mouse per injection or PBS (FIG. 6a). Survival curves (FIG. 6b), tumor luminescence (FIG.6c), and representative IVIS images of the PBT group on days 5 and 30 (FIG. 6d) are shown. FIG.6e- FIG. 6g, Schematic and results of a prophylactic vaccination model for B 16-fLuc-OVA in C57BL / 6 mice. Mice were given three i.m. injections of LNPs containing 10 pg mOVA per injection or PBS, 1 week apart before intrahepatic inoculation of B 16-OVA-fLuc cells on Day 60 (FIG. 6e). Survival curves (FIG. 6f), tumor luminescence (FIG. 6g), and all IVIS images on Day1043985.601_P18418-024 (FIG. 6h) are shown. “PBT,” “MDN,” and “DI-8” stand for PBT mOVA LNPs, MDN mOVA LNPs, and DI-8 mOVA LNPs, respectively. Data represent mean ± s.e.m. with n = 7 biologically independent samples. Survival curves were compared using the log-rank Mantel-Cox test. NS: P > 0.05, *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.

[0039] FIG. 7a, FIG. 7b, FIG. 7c, FIG. 7d, FIG. 7e, FIG. 7f, FIG. 7g, FIG. 7h, FIG. 7i, FIG. 7j, FIG. 7k, and FIG. 71 demonstrate trafficking kinetics of mRNA LNPs following i.m. injection. FIG. 7a-FIG. 7c, Ex vivo quantification of DiR fluorescence (FIG. 7a), Cy5 fluorescence (FIG.7b), and bioluminescence (FIG. 7c) in the injection site versus systemic organs at time points from 0 to 48 h after i.m. injection of DiR-labeled PBT LNPs encapsulating 20 pg Cy5-labeled fLuc mRNA in C57BL / 6 mice. FIG. 7d-FIG. 7f, Ex vivo quantification of DiR fluorescence (FIG. 7d), Cy5 fluorescence (FIG. 7e), and bioluminescence (FIG. 7f) in the injection site, liver, lungs, spleen, local inguinal lymph node (LN), distal inguinal LN, heart, kidneys, and blood. FIG. 7g, Representative in vivo whole-body fluorescence (DiR, Cy5) and bioluminescence (Luc) images of the same mouse at the indicated time points following i.m. injection of DiR- and Cy5-labelled PBT LNPs encapsulating 20 pg fLuc mRNA. FIG. 7h-FIG. 7i, Ex vivo quantification of DiR fluorescence (FIG. 7h) and Cy5 fluorescence (FIG. 7i) in local versus distal inguinal LNs at indicated time points. FIG. 7j, Ex vivo quantification of DiR and Cy5 fluorescence in whole blood at the indicated time points. FIG. 7k-FIG. 71, Flow cytometry analysis showing the percentage of DiR+(FIG. 7k) or Cy5+(FIG. 71) cells among viable circulating cells in the blood. Data represent mean ± s.e.m. with n = 4 biologically independent samples. Group comparisons were performed using two-tailed unpaired t-tests for FIG.7h-FIG. 7i. *P < 0.05, **P < 0.01, ***P < 0.001, ****p <0.0001.

[0040] FIG. 8a, FIG. 8b, FIG. 8c, FIG. 8d, FIG. 8e, FIG. 8f, FIG. 8g, FIG. 8h, FIG. 8i, FIG. 8j, and FIG. 8k show screening of mRNA LNPs for liver-biased transfection following i.m. injection and in vivo assessments of immune activation by selected formulations. FIG. 8a. Schematic overview of the LNP composition library design. The library consisted of the four lipid components shown, with varied ratios of each component as indicated. High-throughput in vitro screening was performed using an automated liquid handler to identify top-performing formulations. FIG. 8b, HepG2 and C2C12 cells were treated with fLuc mRNA LNPs (1 pg / mL). Relative luciferase expression after 24 h incubation in each cell line is plotted, with each dot representing an individual LNP composition (n = 3 replicates). FIG. 8c, Ex vivo liver1143985.601_P18418-02bioluminescence 24 h after i.m. injection of the top 50 formulations grouped into clusters of 10 LNPs each in C57BL / 6 mice. FIG. 8d, Ex vivo bioluminescence in liver, lungs, and spleen 24 h after i.m. injection of the 10 individual formulations within the top cluster, compared with PBT LNPs. FIG. 8e-FIG. 8f, Total bioluminescence flux at the injection site and major organs 24 h post i.m. injection of three selected LNPs in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging using IVIS. Data are expressed as the percentage of total flux at the injection site versus the sum of major organs (FIG. 8e), as well as the percentage expression across individual organs and the injection site (FIG. 8f). FIG. 8g, Ratio of total bioluminescence flux in liver versus muscle injection site for the three selected formulations. FIG. 8h, C57BL / 6 mice were given one i.m. injection of selected LNPs loaded 30 pg mCre per mouse. Mice were sacrificed on day 7 postinjection for analysis. The tdTom+cells were quantified via flow cytometry analysis of the cells isolated from the liver. Percentages of tdTom+cells in the liver are shown. Gating strategies are shown in FIG. 25. FIG. 8i, Titres of OVA-specific IgG antibodies in blood serum on Day 28, determined by ELISA. FIG. 8j-FIG. 8k, C57BL / 6 mice were given three i.m. injections, one week apart, of PBT, MDN, or DL8 LNPs containing 10 pg mOVA per injection or PBS. Mice were sacrificed two weeks after the final injection, and their lymphocytes were isolated from the liver for analysis. The Cellaca MX HACC was employed to count the isolated lymphocytes. Unstimulated lymphocytes isolated from the liver were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and SIINFEKL-H-2Kb tetramer (FIG. 8j), as well as CD44hi, CD62Llo, CD69hi, and CD103+(FIG. 8k). The gating strategy is shown in FIG. 42. Data represent the mean + s.e.m. from a representative experiment (n = 3 biologically independent samples (FIG. 8c-FIG. 8g), n = 4 biologically independent samples (FIG. 8h-FIG. 8k)). Data were analyzed using one-way ANOVA and Tukey’s multiple comparisons test for FIG. 8h-FIG. 8k. For box plots, the box extends from the 25thto the 75thpercentiles with whiskers depicting the minimum / maximum, and the line in the middle of the box is plotted at the median. NS: P > 0.05, *P <0.05, **P <0.01, ***P < 0.001, ****P <0.0001.

[0041] FIG. 9 is representative images of IFN-y-secreting cells from the enzyme-linked immunosorbent assay. Frequency of IFN-y-secreting cells among restimulated splenocytes, assessed via ELISpot. Splenocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).1243985.601_P18418-02

[0042] FIG. 10 shows a gating strategy for flow cytometry plots for T cell responses after vaccination. Initially, lymphocytes isolated from the spleens and livers were selected using SSC-A and FSC-A parameters, followed by singlet selections with SSC-A / SSC-H and FSC-A / FSC-H plots. Viable cells were identified and selected based on the live / dead Fixable Aqua-A and SSC-A plot. Next, the CD3+CD8+T cell population was selected with downstream analysis focused on antigen (OVA)-specific and CD8+T cell subtypes.

[0043] FIG. 11 shows biodistribution and transfection profiles of C10 LNPs following i.v. injection in major organs. LNP biodistribution and transfection at 12 and 24 h post i.v. injection of C10 LNPs (10 pg Cy5-labeled fLuc mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence and bioluminescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency (Cy5) or total bioluminescence flux (Luc) in major organs.

[0044] FIG. 12a and FIG. 12b shows LNP accumulation in kidneys following i.m. versus i.v. injection. Absolute values of total radiant efficiency of Cy5 fluorescence in kidneys at 12 h (FIG.12a) and 24 h (FIG. 12b) following i.m. or i.v. injection of C10 LNPs (10 pg Cy5-labeled fLucmRNA per mouse) in C57BL / 6 mice, quantified via ex vivo fluorescence imaging with IVIS. Datarepresent mean ± s.e.m., n = 4 biologically independent samples. NS, not significant: P > 0.05(two-tailed unpaired t-test).

[0045] FIG. 13 shows LNP transfection efficiency in the liver following i.m. versus i.v. injection. Absolute values of total luminescence flux in the liver at 24 h following i.m. or i.v. injection of C10 LNPs (10 pg fLuc mRNA per mouse) in C57BL / 6 mice, quantified via ex vivobioluminescence imaging with IVIS. Data represent mean ± s.e.m., n = 4 biologically independentsamples.

[0046] FIG. 14a and FIG. 14b show assessment of OT-I cell migration at 24 h post LNP injection. FIG. 14a, Timeline for OT-I cell migration experiment. CD8+ T cells were isolated from OT-I mice and labeled with CellTrace™ Violet (CTV). C57BL / 6 mice were first given one i.m. injection of PBS or C10 LNPs loaded with mOVA (10 pg mOVA per mouse). Six hours post- injection, the same mice were given one i.v. injection (tail vein) of 2.25×106CTV-labelled OT-I CD8+ T cells. Mice were sacrificed 24 or 48 hours post LNP / PBS injection, and their cells were isolated from spleens, livers, and lungs for analysis. FIG. 14b, The Cellaca MX High-throughput Automated Cell Counter was employed to count the cells within the harvested tissues. Isolated cells were assessed via flow cytometry to determine the count of cells positive for CD3, CD8, and CTV in1343985.601_P18418-02each organ at 24 h post LNP injection, c, Biodistribution of CTV-labeled OT-I cells across three major organs in PBS -treated and LNP-treated mice at the 24-h time point. Percentages represent the proportion of OT-I cells from the total number originally injected (2.25X106cells).

[0047] FIG. 15 is representative flow cytometry plots for OT-I migration post LNP injection at the 48-h time point. C57BL / 6 mice were first given one i.m. injection of PBS or C10 LNPs loaded withmOVA (10 pg mOVA per mouse). Six hours post-injection, the same mice were given onei.v. injection (tail vein) of 2.25×106CTV-labelled OT-I CD8+T cells. Mice were sacrificed 48 h post LNP / PBS injection, and their cells were isolated from the spleen, liver, and lungs for analysis. Percentages of cells positive for CTV gated on CD8+cells are shown.

[0048] FIG. 16 shows biodistribution profiles of selected LNP compositions following i.m. injection in injection site vs. major organs. Biodistribution at 12 h post i.m. injection of four selected LNPs (10 pg Cy5-labeled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency at the injection site versus major organs.

[0049] FIG. 17 shows biodistribution profiles of selected LNP compositions following i.m. injection across individual organs. Biodistribution at 12 h post i.m. injection of four selected LNPs (10 pg Cy5-labeled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data are expressed as the percentage of total radiant efficiency across individual organs.

[0050] FIG. 18 shows absolute values of Cy5 fluorescence accumulation across injection site and major organs at the 12-h time point. Absolute values of total radiant efficiency of Cy5 fluorescence measured at 12 h post i.m. injection of four selected LNPs (10 pg Cy5-labelled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data represent mean ± s.e.m., n = 4 biologically independent samples.

[0051] FIG. 19 shows absolute values of Cy5 fluorescence accumulation across injection site and major organs at the 24-h time point. Absolute values of total radiant efficiency of Cy5 fluorescence measured at 24 h post i.m. injection of four selected LNPs (10 pg Cy5-labelled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS. Data represent mean ± s.e.m., n = 4 biologically independent samples.

[0052] FIG. 20 is representative images of ex vivo fluorescence imaging at 12 and 24 h following injection of selected LNP compositions. Representative images of Cy5 fluorescence in injection1443985.601_P18418-02site and major organs measured at 12 and 24 h post i.m. injection of four selected LNPs (10 pg Cy5-labelled mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo fluorescence imaging with IVIS.

[0053] FIG. 21 shows absolute values of total bioluminescence flux across injection site and major organs at the 24-h time point. Absolute values of total bioluminescence flux measured at 24 h post i.m. injection of four selected LNPs (10 pg fLuc mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging with IVIS. Data represent mean ± s.e.m., n = 4 biologically independent samples.

[0054] FIG. 22 shows absolute values of total bioluminescence flux across injection site and major organs at the 48-h time point. Absolute values of total bioluminescence flux measured at 48 h post i.m. injection of four selected LNPs (10 pg fLuc mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging with IVIS. Data represent mean ± s.e.m., n = 4 biologically independent samples.

[0055] FIG. 23 is representative images of ex vivo bioluminescence imaging at 24 and 48 h following injection of selected LNP compositions. Representative images of bioluminescence in injection site and major organs at 12 and 24 h post i.m. injection of four selected LNPs (10 pg fLuc mRNA per mouse) in C57BL / 6 mice, assessed via ex vivo bioluminescence imaging with IVIS.

[0056] FIG. 24 shows Z-average size and PDI of the four selected LNP formulations measured by DLS (n = 3) after dialysis. Data are presented as mean ± S. D. See Table 1 for composition details, as well as zeta potential and encapsulation efficiency of the selected LNP formulations.

[0057] FIG. 25 is the gating strategy for flow cytometry plots for assessment of liver cell transfection by the selected LNP formulations. Initially, cells isolated from the livers were selected using SSC-A and FSC-A parameters, followed by singlet selections with SSC-A / SSC-H and FSC-A / FSC-H plots. Viable cells were identified and selected based on the live / dead Fixable Aqua-A and SSC-A plot. Next, the immune cell (CD45), endothelial cell (CD31), and epithelial cell (CD326) populations were excluded, followed by selection of tdTom+hepatocytes.

[0058] FIG. 26 is the gating strategy for flow cytometry plots for assessment of lung cell transfection by the selected LNP formulations. Initially, cells isolated from the lungs were selected using SSC-A and FSC-A parameters, followed by singlet selections with SSC-A / SSC-H and FSC-A / FSC-H plots. Viable cells were identified and selected based on the live / dead Fixable Aqua-A1543985.601_P18418-02and SSC-A plot. Next, the immune cell (CD45), endothelial cell (CD31), and epithelial cell (CD326) populations were excluded, followed by the selection of tdTom+cells. The tdTomato FMO (Fluorescence Minus One) control for tdTom+cell gating is included as a reference, as the population separation is not distinct.

[0059] FIG. 27 is representative flow cytometry plots for assessments of cell transfection in the liver and lungs by the selected LNP formulations. C57BL / 6 mice were given one i.m. injection of PBT, MDN, or DI-8 LNPs loaded with mCre (30 pg mCre per mouse). Successful mRNA delivery and targeting of the floxed STOP sequence turns on tdTomato expression. Mice were sacrificed on Day 7 post-injection for analysis. The livers and lungs were harvested and homogenized into cell suspension. tdTom+cell percentages within each organ were quantified via flow cytometry. Representative flow cytometry plots for tdTom+hepatocytes in the liver and tdTom+cells in the lungs seven days post-injection are shown. The gating strategies are shown in FIG. 25 and FIG.26.

[0060] FIG. 28a and FIG. 28b show the percentage of tdTom-i- cells in the liver and lungs following LNP injection by different administration routes and volumes. C57BL / 6 mice were given one i.m. injection (100 pL or 50 pL) of PBT LNPs loaded with mCre (30 pg mCre per mouse). Successful mRNA delivery and targeting of the floxed STOP sequence turn on tdTomato expression. Mice were sacrificed on Day 7 post-injection for analysis. Livers and lungs were harvested and homogenized into cell suspension. tdTom+cell percentages within each organ were quantified via flow cytometry. Percentages of tdTom+cells in the liver (FIG. 28a) and lungs (FIG.28b) are shown. The gating strategies are shown in FIG. 25 and FIG. 26.

[0061] FIG. 29 is representative flow cytometry plots for tdTom+ cells in the liver and lungs following LNP injection by different administration routes and volumes. C57BL / 6 mice were given one i.m. injection (100 pL or 50 pL) of PBT LNPs loaded with mCre (30 pg mCre per mouse). Successful mRNA delivery and targeting of the floxed STOP sequence turns on tdTomato expression. Mice were sacrificed on Day 7 post-injection for analysis. Livers and lungs were harvested and homogenized into cell suspension. tdTom+cell percentages within each organ were quantified via flow cytometry. Percentages of tdTom+cells in the liver and lungs are shown. The tdTomato FMO (Fluorescence Minus One) controls for tdTom+transfected cell gating for hepatocytes and lung cells are included as references, as the population separation is not distinct. The gating strategies are shown in FIG. 25 and FIG. 26.1643985.601_P18418-02

[0062] FIG. 30 is the gating strategy for flow cytometry plots for T cell responses after vaccination. Initially, lymphocytes isolated from the liver, lungs, and spleen were selected using SSC-A and FSC-A parameters, followed by singlet selection with FSC-H and FSC-A plot. Viable cells were identified and selected based on the live / dead Fixable Aqua-A staining, and the B220+B cell population was excluded from downstream analysis. Next, CD3+and CD8+cell populations were selected for antigen-specific T cells, in which CD44hlT cells were identified, followed by the subsequent CD62LloCD69hiTRM-like cell identification.

[0063] FIG. 31 is representative images of IFN-y- secreting cells in the liver at the short-term time point (Day 28) from the FluoroSpot assay. Frequency of IFN-y- secreting cells among restimulated lymphocytes assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0064] FIG. 32 is representative images of IFN-y-secreting cells in the lungs at the short-term time point (Day 28) from the FluoroSpot assay. Frequency of IFN-y-secreting cells among restimulated lymphocytes assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0065] FIG. 33 is representative images of IFN-y-secreting cells in the spleen at the short-term time point (Day 28) from the FluoroSpot assay. Frequency of IFN-y-secreting cells among restimulated lymphocytes assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0066] FIG. 34 is representative flow cytometry plots for analysis of CD3+CD8+TNFOH- cells in the spleen at the long-term time point (Day 90). Lymphocytes isolated from the spleen were restimulated in vitro with OVA and SIINFEKL peptide (100 pg / mL OVA and 2 pg / mL SIINFEKL) for 12 h and assessed via intracellular cytokine staining and flow cytometry and to determine the percentages of CD3+CD8+TNF-a+cells.

[0067] FIG. 35 is representative images of IFN-y-secreting cells in the spleen at the long-term time point (Day 90) from the FluoroSpot assay. Frequency of IFN-y-secreting cells among restimulated lymphocytes assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0068] FIG. 36a, FIG. 36b, and FIG. 36c demonstrate antitumor efficacy of the mRNA LNP formulations as therapeutic vaccine in a lung metastasis model. FIG. 36a, Schematic of a therapeutic vaccination model for B16-OVA lung metastatic tumor in C57BL / 6 mice. Mice were1743985.601_P18418-02inoculated via i.v. injection with B16-0VA cells and then given three i.m. injections, 3 days apart, of PBT, MDN, or DI-8 LNPs containing 10 pg mOVA per mouse per injection or PBS. Mice were monitored throughout the treatment schedule for signs of distress and were sacrificed on Day 21 for metastatic foci counting. FIG. 36b, Mouse body weight over time for each group. FIG. 36c, Counted number of metastatic foci in lungs on Day 21. “PBT,” “MDN,” and “DI-8” stand for PBT mOVA LNPs. MDN mOVA LNPs. and DI-8 mOVA LNPs, respectively. Data represent mean + s.e.m. with n = 7 biologically independent samples in (FIG. 36b). For box plots in (FIG.36c), the box extends from the 25thto the 75thpercentiles with whiskers depicting the minimum / maximum, and the line in the middle of the box is plotted at the median (n = 7 biologically independent samples for treatment groups and n = 6 biologically independent samples for PBS group due to early death of one mouse in the PBS group). NS: P > 0.05, **P < 0.01. ***P <0.001.

[0069] FIG. 37 is representative lung images at 21 days after B16-OVA tumor inoculation. Mice were inoculated via intravenous injection with B 16-OVA cells and then given three i.m. injections, 3 days apart, of PBT, MDN, or DI-8 LNPs containing 10 pg mOVA per mouse per injection or PBS. Mice were monitored throughout the treatment schedule for signs of distress and were sacrificed on Day 21 for metastatic foci counting. Black spots indicate the metastatic foci. “PBT,” “MDN,” and “DI-8” stand for PBT mOVA LNPs, MDN mOVA LNPs, and DI-8 mOVA LNPs, respectively, n = 7 biologically independent samples for treatment groups and n = 6 biologically independent samples for PBS group due to early death of one mouse in the PBS group.

[0070] FIG. 38 shows ex vivo quantification of DiR fluorescence, Cy5 fluorescence, and bioluminescence in the injection site versus systemic organs at 1-, 3-, and 6-h time points. Total radiant efficiency of DiR and Cy5 and total bioluminescence flux in in the injection site, liver, lungs, spleen, local inguinal lymph node (LN), distal inguinal LN, heart, kidneys, and blood 1, 3, and 6 hours following i.m. injection of following i.m. injection of DiR-labeled PBT LNPs encapsulating 20 pg Cy5-labeled fLuc mRNA in C57BL / 6 mice. Data represent mean + s.e.m. with n = 4 biologically independent samples.

[0071] FIG. 39 shows ex vivo quantification of DiR fluorescence, Cy5 fluorescence, and bioluminescence in the injection site versus systemic organs at 12-, 24-, and 48-h time points. Total radiant efficiency of DiR and Cy5 and total bioluminescence flux in the injection site, liver, lungs, spleen, local inguinal lymph node (LN), distal inguinal LN, heart, kidneys, and blood 12,1843985.601_P18418-0224, and 48 hours following i.m. injection of DiR-labeled PBT LNPs encapsulating 20 pg Cy5-labeled fLuc mRNA in C57BL / 6 mice. Data represent mean ± s.e.m. with n = 4 biologically independent samples.

[0072] FIG. 40a and FIG. 40b show quantification of luciferase expression in the liver via ex vivo IVIS and ex vivo luciferase assay. C57BL / 6 mice were given one i.m. injection of PBT LNPs encapsulating 20 pg fLuc mRNA. Livers were harvested at the indicated time points for analysis. FIG. 40a, Total bioluminescence flux in the liver at various time points measured via ex vivo IVIS bioluminescence imaging. FIG. 40b, Harvested livers were processed into single-cell suspensions and analyzed using an ex vivo luciferase assay. Relative luciferase units per pg of protein in the liver for each time point are shown. Data represent mean ± s.e.m. with n = 4 biologically independent samples.

[0073] FIG. 41 demonstrate transfection kinetics in local and distal inguinal lymph nodes following i.m. injection. C57BL / 6 mice were given one i.m. injection of PBT LNPs encapsulating 20 pg fLuc mRNA. Local and distal inguinal lymph nodes were harvested at the indicated time points for analysis. Ex vivo quantification of total bioluminescence flux in local versus distal inguinal LNs at the indicated time points is shown. Data represent mean ± s.e.m. with n = 4 biologically independent samples. Group comparisons were performed using two-tailed unpaired t-tests. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001.

[0074] FIG. 42 is the gating strategy for flow cytometry plots for CD 103+ TRM cells after vaccination. Initially, lymphocytes isolated from the liver were selected using SSC-A and FSC-A parameters, followed by singlet selection with SSC-H and SSC-A plot and FSC-H and FSC-A plot. Viable cells were identified and selected based on the live / dead Fixable Aqua-A staining, and the B220+B cell population was excluded from downstream analysis. Next, the CD3+and CD8+cell population was selected for antigen- specific T cells, in which CD44hlT cells were identified, followed by the subsequent CD62LloCD69hiCD103+TRM cell identification.

[0075] FIG. 43a and FIG. 43b show a FluoroSpot assay of IFN-y- secreting lymphocytes in the liver following vaccination. FIG. 43a. Timeline for the short-term vaccination study. C57BL / 6 mice were given three i.m. injections, one week apart, of LNP 10 or PBT LNPs containing 10 pg mOVA per injection or PBS. Mice were sacrificed two weeks after the final injection, and their lymphocytes were isolated from the liver for analysis. FIG. 43b, Frequency of IFN-y-secreting1943985.601_P18418-02cells among restimulated lymphocytes in the liver assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0076] FIG. 44 is representative images of IFN-y- secreting cells in the liver on Day 28 from the FluoroSpot assay. Frequency of IFN-y- secreting cells among restimulated lymphocytes assessed via FluoroSpot. Lymphocytes were restimulated in vitro with SIINFEKL peptide (2 pg / mL SIINFEKL).

[0077] FIG. 45 shows in vitro screening of mRNA lipid nanoparticles for liver cell-specific transfection. Normalized luciferase expression in C2C12 and HepG2 after 24 hr incubation with fLuc mRNA LNPs. Dashed lines represent the 85th percentile. The fourth (bottom right) quadrant pinpoints 50 formulations for subsequent in vivo cluster mode screening.

[0078] FIG. 46A, FIG. 46B, and FIG. 46C show in vivo screening of LNP clusters via i.m. injection to identify LNPs with robust leakage behavior and liver-specific transfection efficiency. FIG. 46A) Transfection efficiency in the injection site and major organs measured by ex vivo bioluminescence of top 50 formulations grouped into clusters of 10 LNPs 24 hrs following i.m. administration (n = 3). FIG. 46B) Luciferase transgene expression percentage by tissue (liver, injection site, and other organs) of the five selected clusters (n = 3). FIG. 46C) Transfection efficiency measured by ex vivo liver bioluminescence of the five selected clusters (n = 3).

[0079] FIG. 47A, FIG. 47B, and FIG. 47C show individual in vivo assessment of the ten formulations in the top cluster to pinpoint compositions with more robust liver preferentially. FIG.47A) Transfection efficiency in the injection site and major organs measured by ex vivo bioluminescence of ten LNP formulations in Cluster 3 and LNP 649 24 hrs following i.m. administration (n = 3). FIG. 47B) Luciferase transgene expression percentage by tissue (liver, injection site, lungs, spleen, kidneys, and heart) of the selected LNP formulations (n = 3). FIG.47C) Transfection efficiency measured by ex vivo liver bioluminescence of the selected LNP formulations (n = 3).

[0080] FIG. 48 is a summary of compositional details of the ten individual LNPs in Cluster 3 and LNP Composition 649. The LNP formulations are grouped into high liver- selective transfection (>80% liver transfection) and low liver- selective transfection (<80% liver transfection) based on percent transfection in the liver. The LNPs are ranked by total bioluminescence in the liver from top to bottom. Molar percentages of the four lipid components (ionizable lipid, helper2043985.601_P18418-02phospholipid, cholesterol, and PEGylated lipids) and N: P ratios are shown for each individual LNP formulation.DETAILED DESCRIPTION

[0081] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. 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. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0082] A. Compositions

[0083] The presently disclosed subject matter provides a composition comprising a lipid nanoparticle (LNP), wherein the LNP comprises an ionizable lipid, a PEGylated lipid, a helper phospholipid, a sterol, and a nucleic acid, the composition of which can be tailored to preferentially localize and transfect cells in specific organs, such as the liver.

[0084] In certain embodiments, the composition comprises a lipid nanoparticle comprising an ionizable lipid; a PEGylated lipid; a helper phospholipid; a cholesterol; and a nucleic acid, wherein: (a) a ratio of the ionizable lipid to the helper phospholipid ranging from about 1 to about 200, including about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125. 130, 135, 140, 145, 150, 155, 160. 165, 170, 175, 180, 185, 190, 195. and 200; (b) a ratio of the cholesterol to the PEGylated lipid ranging from about 10 to about 500, including 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, and 500; (c) a combined percentage of the ionizable lipid and the helper phospholipid ranging from about 20% to about 80%, including about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, and 80%; and (d) a ratio (N / P) of amine groups (N) of the ionizable lipid to phosphate groups (P) of the nucleic acid ranging from about 4 to about 15, including about 4, 5. 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15. hi some embodiments, the presently disclosed LPNs are capable of migrating to one or more systemic organs following intramuscular2143985.601_P18418-02administration at a fraction ranging from about 10% to about 99% of an administered dose, and mediates detectable expression of the nucleic acid in at least one systemic organ including about 10, 15, 20, 25. 30. 35, 40, 45, 50, 55, 60. 65. 70, 75, 80, 85, 90. 95. 96, 97, 98, and 99% of an administered dose.

[0085] In some embodiments: (a) the ionizable lipid has a molar percentage ranging from about 10% to about 60%, including about 10, 15, 20, 25, 30, 35, 40, 45. 50. 55. and 60%; (b) the helper phospholipid has a molar percentage ranging from about 0% to about 40%, including about 0, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, and 40%; (c) the cholesterol has a molar percentage ranging from about 40% to about 90%, including about 45, 50, 55, 60, 65, 70, 75, 80, 85, and 90%; (d) the PEGylated lipid has a molar percentage ranging from about 0.1% to about 5%, including about 0.1, 0.5, 1, 2, 3, 4, and 5%; and (e) an N: P ratio ranging from about 4 to about 15, including about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15.

[0086] In certain embodiments, the lipid nanoparticle exhibits preferential transgene expression localized in the liver. Accordingly, in such embodiments, the lipid nanoparticle comprises: (a) a molar percentage of about 20% to about 30% ionizable lipid, including about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30%; about 20% to about 30% helper phospholipid, including about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and 30%; 40% to about 55% cholesterol, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55%; about 1.5 to about 3.5% PEGylated lipid, including about 1.5, 1.6. 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7. 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, and 3.5%; and an N: P ratio of between about 8 to about 12, including about 8, 9, 10, 11, and 12; (b) a molar percentage of about 10% to about 20% ionizable lipid, including about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20%; about 1% to about 10% helper phospholipid, including about 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%; about 70% to about 90% cholesterol, including about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90%; about 0.1% to about 2% PEGylated lipid, including about 0.1, 0.5. 1.0, 1.1, 1.2, 1.3, 1.4. 1.5, 1.6. 1.7, 1.8. 1.9, and 2.0%; and an N: P ratio of between about 8 to about 12, including about 8, 9, 10, 11, and 12; (c) a molar percentage of about 35% to about 55% ionizable lipid, including about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55; about 5% to about 15% helper phospholipid, including about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15; about 30% to about 50% cholesterol, including about 30, 31, 32, 33, 34, 35, 36. 37, 38, 39, 40, 41, 42, 43, 44, 45. 46. 47, 48, 49, and 50%; about 0.1% to about 2.5% PEGylated lipid, including about 0.1, 0.5, 1.0, 1.1.,2243985.601_P18418-021.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, and 2.5; and an N: P ratio of between about 4 to about 8, including 4, 5, 6, 7, and 8; (d) a molar percentage of about 35% to about 55% ionizable lipid, including about 35, 36, 37. 38. 39. 40. 41. 42. 43. 44.45. 46. 47. 48, 49, 50, 51, 52, 53, 54, and 55; about 0.1% to about 10% helper phospholipid, including about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9. and 10%; about 40% to about 60% cholesterol, including about 40, 41, 42, 43. 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60%; about 0.1% to about 1.0 % PEGylated lipid, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0%; and an N: P ratio of between about 8 to about 10; including about 8, 9 or 10; or (e) a molar percentage of about 35% to about 55% ionizable lipid, including about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, and 55%; about 0.1 to about 10% helper phospholipid, including about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%; about 40% to about 60% cholesterol, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60%; about 0.1% to about 1.0% PEGylated lipid, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0; and an N: P ratio of between about 5 to about 9, including about 5, 6. 7, 8, and 9.

[0087] More particularly, in such embodiments, the lipid nanoparticle comprises: (a) a molar percentage of about 25% ionizable lipid, about 25% helper phospholipid, about 47.5% cholesterol, about 2.5% PEGylated lipid, and an N: P ratio of about 10; (b) a molar percentage of about 14% ionizable lipid, about 6% helper phospholipid, about 79.2% cholesterol, about 0.8% PEGylated lipid, and an N: P ratio of about 10; (c) a molar percentage of about 46.3% ionizable lipid, about 9.4% helper phospholipid, 42.7% cholesterol, about 1.6% PEGylated lipid, and an N: P ratio of about 6; (d) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% cholesterol, about 0.5% PEGylated lipid, and an N: P ratio of about 10; or (e) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% cholesterol, about 0.5% PEGylated lipid, and an N: P ratio of about 7.

[0088] In certain embodiments, the lipid nanoparticle does not exhibit preferential transgene expression localized in the liver. In such embodiments, the lipid nanoparticle comprises: (a) a molar percentage of about 30% to about 40% ionizable lipid, including about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40%; about 10% to about 20% helper phospholipid, including about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20%; about 40% to about 60% cholesterol,, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51. 52. 53. 54, 55. 56, 57, 58, 59, and 60%; about 0.1% to about 1.0% PEGylated lipid, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0%; and an2343985.601_P18418-02N: P ratio of between about 5 and 9, including about 5, 6, 7, 8, and 9; (b) a molar percentage of about 35% to about 55% ionizable lipid, including about 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, and 55%; about 0.1% to about 10% helper phospholipid, including about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%; about 40% to about 60% cholesterol, including about 40, 41, 42, 43, 44, 45. 46, 47, 48, 49, 50, 51, 52, 53. 54, 55, 56, 57, 58, 59, and 60%; about 0.1% to about 0.5% PEGylated lipid, including about 0.1. 0.2, 0.3, 0.4, and 0.5%; and an N: P ratio of between about 5 and about 9; including about 5, 6, 7, 8, and 9; (c) a molar percentage of about 40% to about 50% ionizable lipid, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50%; 0.1% to about 10% helper phospholipid, including about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%; about 40% to about 60% cholesterol, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, and 60%; about 0.1% to about 0.5% PEGylated lipid, including 0.1, 0.2, 0.3, 0.4, and 0.5%; and an N: P ratio of between about 2 to about 6, including 2, 3, 4, 5, and 6; (d) a molar percentage of about 40% to about 50% ionizable lipid, including about 40, 41, 42, 43, 44, 45. 46. 47, 48, 49, and 50%; about 0.1% to about 10% helper phospholipid, including about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10%; about 40% to about 60% cholesterol, including about 40, 41, 42, 43, 44, 45, 46, 47. 48, 49, 50, 51, 52, 53, 54, 55, 56. 57, 58, 59, and 60%; about 0.1% to about 0.5% PEGylated lipid, including about 0.1, 0.2, 0.3, 0.4, and 0.5%; and an N: P ratio of between about 8 to about 10; including about 8, 9, and 10; (e) a molar percentage of about 30% to about 40% ionizable lipid, including about 30, 31, 32, 33. 34, 35, 36, 36, 37, 38, 39, and 40%; about 10% to about 20% helper phospholipid, including about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20; about 40% to about 60% cholesterol, including about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52. 53. 54. 55. 56. 57, 58, 59, and 60%; about 0.1 to about 1.0% PEGylated lipid, including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0%; and an N: P ratio of between about 2 to about 6, including 2, 3, 4, 5. and 6; or (f) a molar percentage of about 60% to about 80% ionizable lipid, including about 60, 61, 62, 63. 64, 65, 66, 67, 68, 69, 70. 71. 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, and 90; about 5% to about 10% helper phospholipid, including about 5, 6, 7, 8, 9, and 10%; about 15% to about 25% cholesterol, including about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25%; about 0.01% to about 0.1% PEGylated lipid, including about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, and 0.1%; and an N: P ratio of between about 8 to about 12, including about 8, 9, 10, 11, and 12.2443985.601_P18418-02

[0089] More particularly, in such embodiments, the lipid nanoparticle comprises: (a) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% cholesterol, 0.5% PEGylated lipid, and an N: P ratio of about 7; (b) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% cholesterol, 0.1% PEGylated lipid, and an N: P ratio of about 7; (c) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% cholesterol, 0.1% PEGylated lipid, and an N: P ratio of about 4; (d) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% cholesterol, 0.1% PEGylated lipid, and an N: P ratio of about 10; (e) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% cholesterol, 0.5% PEGylated lipid, and an N: P ratio of about 4; or (f) a molar percentage of about 72% ionizable lipid, 8% helper phospholipid, 19.96% cholesterol, 0.04% PEGylated lipid, and an N: P ratio of about 10.

[0090] As used herein, the term “ionizable cationic lipid” refers to ionizable lipids that are positively charged at acidic pH to condense 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 ionizable lipids, including, SM-102, ACL-0315, A9, 2,2(8, 8) 4C CH3, LP01, DLin-MC3-DMA, OF-02, A6, and A18-Iso5-2DC18; multi-tail ionizable lipids, including 98N12-5, C12-200, cKK-E12, and 9A1P9; ionizable polymeric lipids, including 7C1 and G0-C14; biodegradable ionizable lipids, including L319, 304013, OF-Deg-Lin, and 306-O12B; and branched tail ionizable lipids, including 3060no and FTT5. Representative ionizable lipids suitable for use with the presently disclosed solid nanoparticles are disclosed, for example, in Han et al., An ionizable lipid toolbox for RNA delivery, Nature Communications, 12:7233 (2021), which is incorporated herein by reference in its entirety.

[0091] Representative lipids are disclosed in U. S. Patent No. 11,229,609 for Compositions and methods for organ specific delivery of nucleic acids, to Cheng et al., published Jan. 25, 2022, which is incorporated herein by reference in its entirety, in particular, col. 3- col. 10, and 46-52.

[0092] Representative ionizable lipids are disclosed in Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078-1094 (2021), which is incorporated herein by reference in its entirety:2543985.601_P18418-02

[0093] The chemical structures of representative ionizable lipids known in the art include.SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate);

[0095] DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminoethyl-dioxolane);

[0096] DLin-KC2-DMA(N, N-dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-l-yl-l,3-dioxolane-4-ethanamine); and2643985.601_P18418-02

[0097] 119-23 (9Z-octadecenoic acid, 6-[[3-(dimethylamino)propyl][(9Z)-l-oxo-9-octadecen-l-yl]amino]-7-oxo-7- (tricyclo [3.3.1.13’7] dec- 1 -ylamino)heptyl ester).

[0098] In particular embodiments, the ionizable lipid comprises ALC-0315.HO X. X x.x ~ O -y J. x..' ~ CH,o

[0099] ALC-315 (4-hydroxybutyl)azanediyl]di(hexane-6,l-diyl) bis(2-hexyldecanoate).

[0100] In some embodiments, the PEGylated lipid comprises dimyristoyl glycerol (DMG)-poly ethyleneglycol (PEG) 2000 (DMG-PEG2000). Other representative PEGylated lipids include, but are not limited to, N-(carbonyl-methoxypolyethyleneglycoln)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEGnwhere n is 350, 500, 750, 1000 or 2000), N-(carbonyl-methoxypolyethyleneglycoln)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEGnwhere n is 350, 500, 750, 1000 or 2000), DSPE-polyglycelin-cyclohexyl-carboxylic acid, DSPE-polyglycelin-2-methylglutar-carboxylic acid, polyethylene glycol-dimyristolglycerol (DMG-PEG), polyethylene glycol-distearoyl glycerol (PEG-DSG), or N-octanoyl-sphingosine-1-{ (succinyl[methoxy(polyethylene glycol)2000] } (C8 PEG2000 Ceramide). In some variations of DMPE-PEGn where n is 350, 500, 750, 1000 or 2000, the PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE-PEG 2,000). In some variations of DSPE-PEGnwhere n is 350, 500, 750, 1000 or 2000, the PEG-lipid is N-(Carbonyl-methoxypolyethyleneglycol 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG 2,000).

[0101] In some embodiments, the PEGylated lipid can be modified to comprise a hydroxyl group on the PEG chain. Accordingly, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl ( — OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In 2743985.601_P18418-02certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain.

[0102] In some embodiments, the helper phospholipid is selected from an anionic phospholipid, a zwitterionic phospholipid, and a cationic phospholipid.

[0103] In certain embodiments, the anionic phospholipid is selected from bis(monooleoylglycero)-phosphate (18BMP), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (18PG), and l,2-dimyristoyl-sn-glycero-3-phosphate (14PA). In particular embodiments, the anionic phospholipid is selected from bis(monooleoylglycero)-phosphate (18BMP), l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (18PG).

[0104] In certain embodiments, the zwitterionic phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl)dimethylammonio)ethyl ethyl phosphate (DOCPe), and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), including DSPC50. In particular embodiments, the zwitterionic phospholipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0105] In certain embodiments, the cationic lipid is selected from Nl-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido) ethyl]-3,4-di[oleyloxy]-benzamide, l,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), O-alkyl phosphatidylcholines, l,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPD), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (14:0 EPC), l,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (16:0 EPC), l,2-distearoyl-sn-glycero-3-ethylphosphocholine (18:0 EPC), l,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1 EPC), l-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (16:0-18:1 EPC), l,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (14:1 EPC), dimethyldioctadecylammonium (DDAB), N-(4-carboxybenzyl)-N, N-dimethyl-2,3-bis(oleoyloxy)propan-l-aminium (DOBAQ), l,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), l,2-dipalmitoyl-3-dimethylammonium-propane (16:0 DAP), l,2-dimyristoyl-3-dimethylammonium-propane (14:0 DAP), l,2-dioleoyl-3-dimethylammonium-propane (DODAP) (18:1 DAP), l,2-dimyristoyl-3-trimethylammonium-propane (14:0 TAP), l,2-dipalmitoyl-3-trimethylammonium-propane (16:0 TAP), l,2-stearoyl-3-trimethylammonium-propane (18:0 TAP), l,2-dioleoyl-3-trimethylammonium-propane (18:1 TAP (DOTAP)), 3B-[N-(N', N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride (DC-Cholesterol-HCl), DC-cholesterol, N4-Cholesteryl-Spermine (GL67), l,2-dioleyloxy-3-dimethylaminopropane2843985.601_P18418-02(DODMA), dimyristoyltrimethylammonium propane (DMTAP), 2,3,-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N, N-dimethyl-l-propane trifluoroacetate (DOSPA), N, N-dioleyl-N, N-dimethylammonium chloride (DODAC), l,2-Dioleoylcarbamyl-3-Dimethylammonium-propane (DOCDAP), 1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), dilauryl(Ci2:o) trimethyl ammonium propane (DLTAP), dioctadecylamidoglycyl spermine (DOGS), DC-Choi. l,2-Dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3[beta]-oxy)-3'-oxapentoxy)-3-dimethyl-l-(ci- s,cis-9',12'-octadecadienoxy) propane (CpLinDMA) and N, N-Dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and l,2-N, N'-Dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and combinations and pharmaceutically acceptable salts thereof.

[0106] In particular embodiments, the cationic phospholipid is selected from l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyl dioctadecyl ammonium (DDAB).

[0107] In other embodiments, the helper phospholipid includes 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine,1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[0108] In some embodiments, the sterol is cholesterol. As used herein, the term “sterols” refers to a subgroup of steroids having a hydroxyl group at the 3-position of the A-ring. Sterols are amphipathic lipids having a polar hydroxyl group on the A ring, whereas the remainder of the aliphatic chain is non-polar. A sterol has the following general structure:2943985.601_P18418-02A B

[00109] HO

[0110] In particular embodiments, the steroid is a cholestane or cholestane derivative. In other embodiments, the steroid is a sterol or a sterol derivative. Representative sterols suitable for use with the presently disclosed LNPs include, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol. stigmasterol, brassicasterol, tomatidine, phytosterols, and combinations thereof. In other embodiments, the presently disclosed LPN comprises a sterol-like component, including, but not limited to, tomatine, ursolic acid, alpha-tocopherol, a hopanoid, and a steroids. In particular embodiments the sterol is cholesterol.

[0111] As used herein, the term “steroid” refers to a compound having a core structure comprising four fused rings, including three six-member cyclohexane rings (annotated as rings A, B, and C) and one five-member cyclopentane ring (annotated as the D ring) as provided in the structure immediately hereinbelow:

[00112]

[0113] The functionality of steroids can be tuned by varying the substituent groups on the four-ring core, including, for example, one or more substituent groups selected from alkyl, alkoxyl, hydroxyl, oxo, acyl, and by the oxidation state of the rings. Steroids also can be modified by changing the ring structure, for example by cleaving one of the rings.

[0114] As used herein, the term “nucleic acid” refers to one or more of the following biomolecules, including, but small interfering ribonucleic acid (siRNA), a messenger RNA (mRNA), a micro-ribonucleic acid (miRNA), a primary micro-ribonucleic acid (pri-miRNA), a messenger ribonucleic acid (mRNA), a clustered regularly interspaced short palindromic repeats (CRISPR) related nucleic acid, a CRISPR-RNA (crRNA), a single guide ribonucleic acid (sgRNA), a transactivating 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 acid3043985.601_P18418-02(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 particular embodiments, the nucleic acid comprises plasmid DNA (pDNA) or siRNA. In certain embodiments, the nucleic acid is plasma DNA. In certain embodiments, the nucleic acid comprises siRNA. In yet more certain embodiments, the nucleic acid comprises a combination of pDNA and siRNA. In particular embodiments, the siRNA is an anti-inflammatory siRNA.

[0115] In certain embodiments, the nucleic acid is selected from an antisense oligonucleotide, cDNA, genomic DNA, guide RNA, plasmid DNA (pDNA), vector DNA, mRNA, miRNA, piRNA, shRNA, and siRNA. In particular embodiments, the nucleic acid is mRNA.

[0116] In some embodiments, the nucleic acid comprises a polynucleotide encoding a Cas nuclease. In certain embodiments, the Cas nuclease is selected from Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Casio, Csyl, Csy2, Csy3, Csel, Cse2, Csd, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4. In particular embodiments, the Cas nuclease comprises Cas9.

[0117] In certain embodiments, the lipid nanoparticle further comprises a small guide RNA or a DNA encoding a small guide RNA.

[0118] In certain embodiments, the lipid nanoparticle comprises a vaccine.

[0119] In some embodiments, the lipid nanoparticles described herein have a diameter from about 1 nm to about 100 nm such as, but not limited to, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm,3143985.601_P18418-02about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm.

[0120] In some embodiments, the lipid nanoparticles described herein have a diameter from about 10 to 500 nm. In some embodiments, the lipid nanoparticle can have a diameter greater than 100 nm, greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm or greater than 1000 nm.

[0121] B. Pharmaceutical Formulations

[0122] The LNP compositions may also be formulated as pharmaceutical compositions. Pharmaceutical compositions can optionally comprise one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions can be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed„ Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety). In some embodiments, compositions are administered to humans, human patients or subjects. The phrase “active ingredient” generally refers to polynucleotides to be delivered as described herein.

[0123] Formulations and pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0124] A pharmaceutical composition in accordance with the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition3243985.601_P18418-02comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0125] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure can vary, depending upon the identity, size, and / or condition of the subject being treated and further depending upon the route by which the composition is to be administered.

[0126] In some embodiments, the compositions and formulations described herein can contain at least one LNP. As a non-limiting example, the composition can contain 1. 2, 3, 4 or 5 LNPs. In some embodiments, the compositions described herein can comprise more than one type of LNP.

[0127] Although the descriptions of pharmaceutical compositions and formulations provided herein are principally directed to pharmaceutical compositions and formulations that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g., non¬ human mammals.

[0128] The present disclosure provides pharmaceutical formulations that comprise an LNP described herein. The LNPs described herein can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) pennit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide); (4) alter the biodistribution (e.g., target the LNP to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and / or (6) alter the release profile of encoded protein in vivo.

[0129] A pharmaceutically acceptable excipient, as used herein, includes, but are not limited to, any and all solvents, dispersion media, or other liquid vehicles, dispersion or suspension aids, diluents, granulating and / or dispersing agents, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, binders, lubricants or oil, coloring, sweetening or flavoring agents, stabilizers, antioxidants, antimicrobial or antifungal agents, osmolality adjusting agents, pH adjusting agents, buffers, chelants, cyoprotectants, and / or bulking agents, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science3343985.601_P18418-02and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott. Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety).

[0130] Exemplary diluents include, but are not limited to, calcium or sodium carbonate, calcium phosphate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, etc., and / or combinations thereof.

[0131] Exemplary granulating and / or dispersing agents include, but are not limited to, starches, pregelatinized starches, or microcrystalline starch, alginic acid, guar gum, agar, polyvinyl¬ pyrrolidone), (providone), cross-linked poly(viny 1-py rrolidone) (crospo vidone), cellulose, methylcellulose, carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, etc., and / or combinati ons thereof,

[0132] Exemplary surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat. cholesterol, wax. and lecithin), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], glyceryl monooleate, polyoxyethylene esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers (e.g.. polyoxyethylene lauryl ether [BRIJ®30]), PLUOR1NC®F 68, POLOXAMER®! 88, etc. and / or combinations thereof.

[0133] Exemplary binding agents include, but are not limited to. starch, gelatin, sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol), amino acids (e.g.. glycine), natural and synthetic gums (e.g., acacia, sodium alginate), ethylcellulose, hydroxyethylcellulose, hydroxypropyl methylcellulose, etc., and combinations thereof.

[0134] Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, rn-cresol, methionine, butylated hydroxytoluene, monothioglycerol, sodium or potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, etc., and combinations thereof.

[0135] Exemplary chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate. fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, trisodium edetate, etc., and combinations thereof.

[0136] Exemplary antimicrobial or antifungal agents include, but are not limited to, benzalkonium chloride, benzethonium chloride, methyl paraben, ethyl paraben, propyl paraben, butyl paraben,3443985.601_P18418-02benzoic acid, hydroxybenzoic acid, potassium or sodium benzoate, potassium or sodium sorbate, sodium propionate, sorbic acid. etc., and combinations thereof.

[0137] Exemplary preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, ascorbic acid, butylated hydroxyanisol. ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), etc., and combinations thereof.

[0138] In some embodiments, the pH of polynucleotide solutions is maintained between pH 5 and pH 8 to improve stability. Exemplary buffers to control pH can include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine- HC1), sodium malate, sodium carbonate, etc., and / or combinations thereof.

[0139] Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium or magnesium lauryl sulfate, etc., and combinations thereof.

[0140] The pharmaceutical composition described here can contain a cryoprotectant to stabilize a polynucleotide described herein during freezing. Exemplary cryoprotectants include, but are not limited to mannitol, sucrose, trehalose, lactose, glycerol, dextrose, etc., and combinations thereof.

[0141] The pharmaceutical composition described here can contain a bulking agent in lyophilized polynucleotide formulations to yield a “pharmaceutically elegant” cake, stabilize the lyophilized polynucleotides during long, term (e.g., 36 month) storage. Exemplary bulking agents can include, but are not limited to sucrose, trehalose, mannitol, glycine, lactose, raffinose, and combinations thereof.

[0142] The composition may also comprise a free reducing agent or antioxidant. Exemplary free reducing agents or antioxidants include, but are not limited to, potassium metabisulfite, sodium thioglycolate, tris(2-carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione. dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, and lipoic acid.

[0143] In some embodiments, the presently disclosed compositions can be formulated for delivery to the mucous. In such embodiments, the LNPs can be engineered to alter the surface properties of particles so that the lipid nanoparticles can penetrate the mucosal barrier as described in U. S. Pat. No. 8,241,670 or Inti. Pub. No. WO2013110028. each of which is herein incorporated by reference in its entirety.3543985.601_P18418-02

[0144] The LNPs engineered to penetrate mucus can comprise a polymeric material (e.g., a polymeric core) and / or a polymer-vitamin conjugate and / or a tri-block co-polymer. The polymeric material can include, but is not limited io, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, polystyrenes), polyimides, polysulfones, polyurethanes, poly acetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates.

[0145] LNPs engineered to penetrate mucus also can include surface altering agents such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., -acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin 34 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.

[0146] In some embodiments, the mucus penetrating LNP can be a hypotonic formulation comprising a mucosal penetration enhancing coating. The formulation can be hypotonic for the epithelium to which it is being delivered. Non-limiting examples of hypotonic formulations can be found in, e.g.. Inti. Pub. No. WO2013110028, herein incorporated by reference in its entirety.

[0147] C. Methods of Treatment

[0148] In some embodiments, the presently disclosed subject matter provides a method for treating a disease, condition, or disorder, the method comprising administering a composition described herein, or a vaccine comprising the composition, to a subject in need of treatment thereof.

[0149] In certain embodiments, administration of the composition or the vaccine elicits a specific T cell response and / or cellular immunity. Without wishing to be bound to any one particular theory, it is thought that the substantial leakage of the presently disclosed LNPs leads to protein expression in peripheral tissues, which will recruit T cells to these tissues and become tissueresident T cells. These cells offer stronger immune protection to clear the invading cells, such as cancer cells. They also provide longer-term protection, i.e., stronger immune memory. In certain embodiments, the presently disclosed method for delivering a vaccine can be used for cancer treatment to clear invading cancer cells, thus preventing metastasis or preventing cell engraftment.3643985.601_P18418-02

[0150] In certain embodiments, administration of the composition or the vaccine elicits a tissuespecific immune response at the primary site of infection.

[0151] In certain embodiments, the disease, condition, or disorder comprises a disease, condition, or disorder of the lung. In particular embodiments, the disease, condition, or disorder of the lung is selected from pneumonia, respiratory syncytial virus (RSV), influenza, tuberculosis, and a coronavirus.

[0152] In certain embodiments, the disease, condition, or disorder comprises a cancer. In certain embodiments, administering the composition or the vaccine prevents or reduces one or more cancer cells remaining from a post-tumor excision or other surgical cancer treatment from metastasizing to one or more other organs.

[0153] In certain embodiments, the disease, condition, or disorder comprises an infectious disease. In particular embodiments, the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the liver. In more particular embodiments, the disease, condition, or disorder of the liver is selected from malaria, hepatitis B, and hepatitis C.

[0154] In certain embodiments, administration of the composition or the vaccine comprising presently disclosed LNPs having a strong leakage property achieves gene expression in selected peripheral tissues and a lower systemic toxicity.

[0155] In certain embodiments, the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the lymphatic system. In particular embodiments, the disease, condition, or disorder of the lymphatic system comprises human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS). In certain embodiments, tissue-resident T cells clear pathogen-invading cells, such as in methods for treating or preventing malaria or HIV.

[0156] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of. preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0157] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are3743985.601_P18418-02effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates. e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0158] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0159] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a composition disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage3843985.601_P18418-02forms that are administered alternately or sequentially on the same or separate days. Tn one embodiment of the presently disclosed subject matter, 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., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0160] Further, the compositions disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compositions, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0161] The timing of administration of a composition disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a composition described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a composition described herein and at least one additional therapeutic agent can receive a composition and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0162] When administered sequentially, the agents can be administered within 1, 5, 10, 30. 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the composition described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either a composition or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0163] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents3943985.601_P18418-02relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0164] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a composition described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0165] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0166] QS / QA+ QH / QB= Synergy Index (SI)

[0167] wherein:

[0168] Q is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0169] Qais the concentration of component A, in a mixture, which produced an end point;

[0170] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0171] Qb is the concentration of component B, in a mixture, which produced an end point.

[0172] Generally, when the sum of QS / QA and QB / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0173] D. Forms of Administration

[0174] In certain embodiments, the administering is via intramuscular (i.m.) injection. In other embodiments, the administering is by or subcutaneous (s.c.) injection.4043985.601_P18418-02

[0175] The compositions described above can be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalational, buccal, sublingual, intrapleural, intrathecal, intranasal, and the like. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), and transmucosal administration. In some embodiments, compositions can be administered in a way that allows them cross the blood -brain barrier, vascular barrier, or other epithelial barrier. In some embodiments, a formulation for a route of administration can include at least one inactive ingredient.

[0176] The compositions can be formulated using the methods described herein. The compositions can contain polynucleotides that can be modified and / or unmodified. The compositions can further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The compositions can be delivered to the cell using routes of administration known in the art and described herein.

[0177] A pharmaceutical composition for parenteral administration can comprise at least one inactive ingredient. Any or none of the inactive ingredients used can have been approved by the US Food and Drug Administration (FDA). A non-exhaustive list of inactive ingredients for use in pharmaceutical compositions for parenteral administration includes hydrochloric acid, mannitol, nitrogen, sodium acetate, sodium chloride, and sodium hydroxide.

[0178] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions can be formulated according to the known art using suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution, U. S. P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables. The sterile formulation can also comprise adjuvants such as local anesthetics, preservatives, and buffering agents.4143985.601_P18418-02

[0179] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0180] Injectable formulations can be for direct injection into a region of a tissue, organ and / or subject. As a non-limiting example, a tissue, organ and / or subject can be directly injected a formulation by intramyocardial injection into the ischemic region. (See. e.g., Zangi et al. Nature Biotechnology 2013, which is herein incorporated by reference in its entirety).

[0181] To prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This can be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms can be made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0182] Unless otherwise noted, the chemical terms provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the " Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997). The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / -2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 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.4243985.601_P18418-02

[0183] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0184] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of.” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0185] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.

[0186] EXAMPLE 1

[0187] Systemic trafficking of mRNA lipid nanoparticle vaccine following intramuscular injection generates potent tissue-specific T cell response

[0188] Overview

[0189] The mRNA lipid nanoparticles (LNPs) have been designed as a new generation of vaccine carriers to elicit potent immune responses against infectious diseases and cancer. Despite the clinical success and rapid advancements in mRNA LNP technologies, the trafficking patterns of4343985.601_P18418-02LNPs after intramuscular (i.m.) administration and the subsequent tissue-specific immunological effects have not been systematically characterized. This Example demonstrates that trafficking of mRNA LNPs to different organs following i.m. injection is crucial for the induction of tissuespecific immunity beyond systemic immune response, particularly in tissue-resident CD8+T cell generation, which is important to localized defense. By fine-tuning the composition of mRNA LNPs, trafficking patterns to systemic organs can be modulated, which can alter the resulting tissue-specific immune response. Formulations with a greater ability to enter the bloodstream can preferentially localize and transfect cells in specific organs like the liver, elicit stronger tissuespecific CD8+T cell immune responses, and achieve enhanced efficacy in a liver tumor model. These findings highlight the potential to tailor mRNA LNP compositions to modulate trafficking following i.m. injection, thereby providing novel strategies for designing tissue-specific vaccines. Such strategies are particularly valuable for organ-specific diseases like cancer and infectious diseases, where tissue targeting and long-lasting immunity are essential for therapeutic success.

[0190] Background

[0191] Lipid nanoparticles (LNPs) now serve as widely used COVID-19 vaccine delivery platforms for antigen-encoding mRNA and hold promise for additional prophylactic and therapeutic applications. Hou et al., 2021; Gebre et al., 2021; Kowalski et al., 2019. Recent studies demonstrated that the composition of mRNA LNP formulations impacts their pharmacokinetics. Safford et al., 2024; Lokugamage et al., 2021; Cheng et al., 2020; Oberli et al., 2017; Li et al., 2015. The biodistribution and transfection behavior of LNPs administered by various routes — such as i.m., i.v., and subcutaneous (s.c.) injections — can be engineered by modifying their composition, thereby achieving tissue- and cell-specific transgene expression. Zhu et al., 2022; Safford et al., 2024; Lokugamage et al., 2021; Cheng et al., 2020; Oberli et al., 2017; Li et al., 2015; Zhu et al., 2024a; Xue et al., 2022; Patel et al., 2019; Zhu et al., 2024b.

[0192] Additionally, adjusting LNP composition can influence the profile of transfected cell types, altering immune activation patterns, which ultimately impact vaccination outcomes. Zhu et al., 2024; Dobrowolski et al., 2022; Patel et al., 2022. On the other hand, an increasing number of recent studies have reported that a portion of certain LNPs administered via i.m. injection enters the systemic circulation and accumulates in major organs like the liver and lungs. Pateev et al., 2024; Ma et al., 2024; Di et al., 2022; Hassett et al., 2024. The therapeutic implications of LNP4443985.601_P18418-02trafficking and unintended expression in non-lymphoid organs, however, remain poorly understood.

[0193] Scope

[0194] In this Example, without wishing to be bound to any one particular theory, it was thought that tissue-specific CD8+T cell immune responses could be generated by optimizing LNP compositions to promote preferential entry and expression in different non-lymphoid organs, including the liver and lungs (FIG. la). We first investigated the trafficking of mRNA LNPs following i.m. injection, focusing on their systemic entry and retention in non-lymphoid tissues, as well as the resulting systemic and tissue- specific immunological outcomes. We then examined the pathways underlying tissue-specific immune responses generated by i.m. injection of mRNA LNPs and their relationships with LNP trafficking and tissue- specific antigen expression. By further assessing the biodistribution profiles of different mRNA LNP compositions, we identified formulations with a higher degree of expression in either the liver or lungs, demonstrating the ability to modulate LNP trafficking and local expression by adjusting LNP compositions. These formulations were further evaluated for their short- and long-term immune profiles, including systemic cellular and humoral immune responses, as well as tissue- specific CD8+T cell activation and CD8+TRM cell generation. Finally, we revealed the feasibility of tuning mRNA LNP formulation to induce durable systemic and localized immunity with improved therapeutic and prophylactic outcomes using mouse liver cancer models.

[0195] Results

[0196] Tissue-specific T cell responses following i.m. injection of mRNA LNPs

[0197] Given the limited understanding of mRNA LNP vaccine-mediated T cell responses at the tissue level, we first aimed to examine both systemic and tissue-specific immune responses induced by an LNP formulation (C10 mRNA LNP) recently developed for anti-tumor immunity through systemic Th1 and Th2 immune modulation, Zhu et al., 2024b, with a particular focus on the CD8+T cell responses in the liver. C57BL / 6 mice were administered C10 LNPs containing 10 μg ovalbumin mRNA (mOVA) by i.m. injection in the right quadriceps muscle on Days 0, 21, and 42. On Day 70, we observed a significant increase in the number of IFN-y-secreting lymphocytes (P = 0.0179) isolated from the spleens of LNP-treated mice compared to the controls (FIG. lb, FIG. 9). Additionally, a marked elevation in the number of OVA-specific CD8+T cells was detected in the spleens for the LNP-treated group, showing a 21 -fold increase compared with the4543985.601_P18418-02PBS control (FIG. 1c). Moreover, the CIO mOVA LNPs induced a significantly higher anti-OVA IgG titer (average of 1:107, P = 0.0010) than the PBS group, indicating a strong humoral response (FIG. Id). These data confirm that CIO mOVA LNPs can elicit potent and sustained systemic immunity over an extended vaccination period, providing prolonged immune protection.

[0198] We next evaluated tissue-specific immunity induced by the CIO LNPs following i.m. injections. Interestingly, in addition to the systemic immunity, a notable increase in IFN-y-secreting lymphocytes (P = 0.0262) and a 10-fold higher frequency of OVA-specific CD8+T cells were detected in the liver of LNP-treated mice compared to the PBS group (FIG. le-FIG. 1g). Furthermore, a substantial population of CD8+effector memory T (TEM) cells (average of 5 × 104, P = 0.0007 compared to the PBS group) was observed in the liver, indicating the potential of CD8+TRMcell formation (FIG. 1h). Previous studies have predominantly reported local transfection at the injection site and systemic immunity, with limited focus on liver-specific immune responses following i.m. delivery. Zhu et al., 2024b. In this experiment, a tissue-specific T cell response was observed in a non-lymphoid organ, suggesting the potential of using these T cells for rapid and effective immune responses at the critical site of pathogen entry. We then further investigated the mechanism underlying the generation of local immunity following i.m. administration of mRNA LNP vaccines.

[0199] Systemic LNP trafficking results in tissue-specific antigen expression and subsequent T-cell recruitment

[0200] To examine the trafficking profile of LNPs following i.m. injection, we administered C10 LNPs encapsulating 10 μg Cy-5-labeled mRNA into the right quadriceps of C57BL / 6 mice and monitored their biodistribution at 12 and 24 h post-injection. At both time points, LNPs were detected in the injection site and major organs, indicating trafficking from the injection site to the circulation (FIG. 2a, FIG. 2b). Notably, at the 24-h time point, most LNPs (51.7%) that travelled out of the injection site were found in the liver, suggesting a biased trafficking pattern. Interestingly, kidneys exhibited relatively high signal percentages (52.2% at 12 h and 26.3% at 24 h), which are markedly greater than those observed after i.v. administration (FIG. 11). When comparing absolute fluorescence values, however, kidney Cy5 levels were similar between i.m. and i.v. routes (FIG. 12). The higher percentages observed after i.m. dosing reflect normalization to a lower total organ signal (i.e., a smaller denominator), rather than a disproportionate increase in kidney-associated fluorescence after i.m. injection.4643985.601_P18418-02

[0201] To determine if LNP transfection occurred at the sites where they trafficked, C57BL / 6 mice were injected with 10 pg of firefly luciferase-encoding mRNA (mLuc) LNPs intramuscularly, and the luciferase protein expression levels at the injection site and in major organs were measured at 24 and 48 h post-injection. As shown in FIG. 2c, FIG. 2d, a substantial portion of luciferase expression was found in major organs (58.1%) in addition to the local injected muscle (41.9%). Among the transfected tissues, liver accounted for 50.0% and 58.8% of expression at 24-h and 48-h time points, respectively, consistent with the biodistribution profile. Thus, i.m.-injected CIO LNPs trafficked in part to the liver and result in considerable mRNA-encoded protein expression in the liver. Consistent with what was observed with biodistribution, the transfection profile following i.m. injection also differs from that observed with i.v. administration, with markedly reduced kidney transfection and greater hepatic expression (approximately 100-fold higher in transgene expression in the liver than i.m. administration) (FIGS. 11-13).

[0202] To validate the causal relationship between LNP trafficking and formation of liver-specific immunity, the third i.m. injection in the vaccination schedule was replaced with an i.v. injection to directly introduce LNPs into the bloodstream and further enhance liver expression (FIG. 2e). As illustrated in FIG. 2f-FIG. 2i, mice that received an i.v. injection of CIO mOVA LNPs as the third dose exhibited a 6.0-fold increase in the numbers of CD8+T cells and TEMcells, as well as a 5.2-fold increase in the frequency of IFN-y- secreting lymphocytes in the liver, compared to mice that received three doses by i.m. injection. These data collectively suggest that a higher degree of antigen expression in the liver leads to an elevated level of tissue-specific cellular immune response, supporting the role of LNP trafficking following i.m. injection in generating localized immune responses within the liver.

[0203] Our results showed that a substantial portion of LNPs can traffic to the liver after i.m. injection, mediate antigen expression, and enriched antigen-specific CD8+T cells including CD8+TRM cells in the liver. To further elucidate the connection between LNP-mediated transfection and the recruitment of immune cells, we explored if the accumulation of OVA-specific T cells in the liver was driven by tissue-specific antigen expression. We injected and monitored migration of OT-I cells 6 h after i.m. injection of LNPs. OT-I cells are CD8+T cells expressing a transgenic T cell receptor specific for the SIINFEKL peptide epitope. C57BL / 6 mice were first given (i.m.) C10 LNPs containing 10 μg of mOVA. Six hours post-injection, 2.25×106CellTrace Violet (CTV)-labeled OT-I cells were introduced into mice i.v., and the distribution of OT-I cells was4743985.601_P18418-02subsequently monitored at the 24-h and 48-h time points (FIG. 2j). At the 24-h time point, the difference in OT-I cell migration was minor between LNP-treated and PBS-treated groups, with a slight increase in the number of OT-I cells in the liver of the LNP-treated mice (FIG. 14). By 48 h, however, there was a more than 6.0-fold elevation (P = 0.01) in the number of OT-I cells in the liver of LNP-treated mice compared to the PBS-treated group, while the number of OT-I cells in other organs remained unchanged (FIG. 2k, FIG. 15). As depicted in FIG. 21, only 3.1% of the total number of injected OT-I cells were detected in the liver in the PBS-treated group. In contrast, the liver had the highest percentage of OT-I cells among the three organs examined in the LNP-treated group, accounting for 25.4% of the total injected OT-I cells. These findings suggest that T cells are being recruited to the liver, presumably in response to liver-specific expression of antigen induced by mRNA LNPs that trafficked from the i.m. injection site to the liver.

[0204] LNP trafficking and transfection of major organs is LNP composition -dependent

[0205] We next determined whether LNP trafficking and transfection in major organs vary among different LNP formulations. We selected three LNP compositions based on FDA-approved formulations: MDN (used in the Modema COVID-19 vaccine), Baden et al., 2021. PBT (using the ALC-0315 as the ionizable lipid and same composition as in the Pfizer-BioNTech COVID-19 vaccine, Polack et al., 2020, except DMG-PEG2000 as the PEGylated lipid), and ALN (used in the Alnylam Onpattro formulation), Adams et al., 2018, in addition to one LNP formulation DL8, which was described in our previous report showing high levels of gene expression in the liver through i.v. injections. Zhu et al., 2022.

[0206] The biodistribution profiles of these selected LNP formulations were first evaluated. C57BL / 6 mice received one i.m. injection of LNPs containing 10 μg of Cy-5-labelled mRNA at their right quadriceps; and the biodistribution of the LNPs was monitored at 12-h and 24-h time points (Fig 3a). Trafficking was observed beyond the injection site for all formulations. As depicted in FIG. 3a, distinct trafficking patterns were observed amongst the selected formulations. For instance, the DL8 exhibited 13.9% “leakage” from the injection site into the systemic circulation, whereas the MDN LNPs yielded a much higher leakage of 31.4% (P < 0.0001). Despite the differences in leakage, once the LNPs entered the bloodstream, they displayed comparable biodistribution patterns across major organs with approximately 40% to the liver, approximately 10% to the lungs, and approximately 30% to the spleen (FIG. 3b, FIGS. 16-20).4843985.601_P18418-02

[0207] Next, we evaluated the transfection efficiency of the selected LNP formulations in different tissues. Each C57BL / 6 mouse received one i.m. injection of LNPs containing 10 μg of mLuc at the right quadriceps; and the luciferase expression levels at the injection site and the major organs were measured at 24- and 48-h post-injection. As depicted in FIG. 3c, different LNP formulations exhibited distinct transfection patterns outside of the injection site. Despite similar biodistribution patterns following systemic trafficking, these LNPs showed different transfections in major organs, independent of the biodistribution pattern (FIG. 3d). The total luminescence intensity measurements revealed that the PBT LNPs achieved the highest level of mLuc expression in the liver (P = 0.0377 compared with DL8). (FIG. 3e). Meanwhile, the MDN LNPs exhibited the highest level of absolute mLuc expression level in the lungs compared to the other formulations (P = 0.0017 compared with DI-8) (FIG. 3f). In contrast, the DI-8 showed minimal transfection in systemic organs, with the majority of transgene expression localized at the injection site (FIG. 3e-FIG. 3g). Notably, although DI-8 appears to have high relative percentages of liver and lung transfection (40.8% and 21.2%, respectively) in FIG. 3d, these values reflect proportional distribution and do not capture the total transgene expression levels across all organs. As shown by the absolute values, following i.m. injection, a substantial fraction of DI-8 LNPs failed to achieve effective cellular transfection, whereas PBT and MDN produced significantly higher absolute expression in the liver and lungs, respectively (FIG. 3e-FIG. 3f, FIGS. 21-23). Thus, while FIG. 3d illustrates relative biodistribution patterns, the absolute values provide a more accurate measure of transfection efficiency.

[0208] Although these LNP compositions share similar physical properties such as size, PDI, zeta potential, and encapsulation efficiency (FIG. 24, Tables 1 and 2), they exhibited different tissuespecific transfection profiles following systemic trafficking from the i.m. injection site. Based on these results, we selected PBT, MDN, and DI-8 LNPs as representative formulations with biased tissue-specific expressions in the liver, lungs, and no systemic organ transfection, respectively.Table 1. Composition details and characterization of the five evaluated LNP formulations.Formulation Code PBT C10 DI-8 ALN MDN Composition (molar ratio):Ionizable lipid 46.3 40.0 36.36 49.74 50.04943985.601_P18418-02Helper lipid 9.4 40.0 3.64 10.26 10.0 Cholesterol 42.7 19.96 59.88 38.46 38.5PEG2000 1.6 0.04 0.12 1.54 1.5 N / P ratio 6 4 8 4 6 Formulation features:Z-average diameter (nm)126.4 ± 0.6 162.9 ± 2.6 125.6 ± 0.6 126.6 ± 0.9 117.6 ± 1.0Average PDI 0.212 ± 0.018 0.209 ± 0.009 0.181 ± 0.012 0.208 ± 0.022 0.115 ± 0.028 Average Zeta potential (mV) -2.92 ± 0.20 -11.0 ± 2.30 -1.25 ± 0.50 -2.95 ± 0.51 -2.80 ± 0.71Average EE% 96.45 99.03 99.96 99.95 99.85Table 2. Composition details and characterization of LNP Formulations 10 and 61Formulation Code 10 61Composition (molar ratio):Ionizable lipid 25 72Helper lipid 25 8Cholesterol 47.5 19.96DMG-PEG2000 2.5 0.04Formulation features:N / P ratio 10 10Z-average diameter (nm) 84.89 ± 0.63 101.4 ± 1.2(nm)Average PDI 0.147 ± 0.089 0.209 ± 0.012 Average Zeta potential (mV) -14.5 ± 4 -10.8 ± 1.7(mV)5043985.601_P18418-02Average EE% 95.2 89.9

[0209] To further assess the organ-specific trafficking and transfection, we gave one i.m. injection of Cre-recombinase mRNA (mCre) LNPs to genetically engineered tdTomato (tdTom) reporter (Ai9) mice that contain a LoxP-flanked stop cassette that prevents expression of the tdTom protein. This mouse model enables the identification of transfected cells through Cre-recombinase expression, which removes the stop cassette, allowing the fluorescent tdTom protein to be expressed (FIG. 3h). Seven days following i.m. injection, the PBT LNP formulation resulted in the highest percentage, 39.3% (P < 0.0001) of tdTomato-positive (tdTom+) cells in the liver compared to the MDN LNPs (5.4%) and DI-8 LNPs (0.01%) (FIG. 3i, FIG. 3j, FIG. 27). In the lungs, there were no significant differences in transfection efficiency across the three LNP formulations, as all three exhibited relatively low levels of tdTom+cells (FIG. 3k, FIG. 27).

[0210] To explore whether injection volume will alter the extent and distribution of the LNP trafficking following i.m. injection, we evaluated the gene expression efficiency in major organs with different i.m. injection volumes. As shown in FIG. 28 and FIG. 29, changing the i.m. injection volume from 50 to 100 pL did not affect the percentage of tdTom+hepatocytes in the liver, indicating that the observed LNP trafficking was driven by intrinsic LNP properties rather than diffusion kinetics that may be influenced by the injection volume (FIG. 28 and FIG. 29). This reaffirms the importance of LNP composition in modulating the extent and pattern of the LNP trafficking outside the injection site for organ-targeted applications.

[0211] Assessment of short-term systemic and tissue-specific immune activation by selected mRNA LNP formulations following i.m. injection

[0212] We next investigated the effect of biased LNP trafficking and expression in different organs on the generation of tissue-specific immunity. We evaluated short-term immune responses following a prime-and-boost vaccination regimen using the PBT, MDN, and DI-8 LNP formulations, with OVA as the model antigen (FIG. 4a). On Day 28, the frequency of OVA-specific CD8+T cells in the liver of mice treated with PBT mOVA LNPs was 4.3-, 8.4-, and 28.5-fold higher (P < 0.0001) than that of mice treated with the MDN mOVA LNPs, DI-8 mOVA LNPs, and PBS, respectively (FIG. 4b, FIG. 4c). A substantial population of OVA-specific CD8+CD44hiCD62LloCD69hiTRM-like cells was detected in the liver for the PBT mOVA LNP-treated group, with the number being 4.6-, 3.9-, and 22.7-fold higher (P = 0.0002, P = 0.0001, and5143985.601_P18418-02P < 0.0001, respectively) than the MDN mOVA LNP-, DT-8 mOVA LNP-, and PBS-treated groups, respectively (FIG. 4d). This indicates successful induction of tissue- specific immunity and establishment of TRM cells in the liver with the PBT LNPs, which are crucial for long-term protection. In the lungs, there were 2.3-, 9.6-, and 48.3-fold increases (P = 0.0064, P < 0.0001, and P < 0.0001, respectively) in OVA-specific CD8+T cell frequencies after treatment with MDN mOVA LNPs, compared with PBT mOVA LNPs. DI-8 mOVA LNPs, and PBS, respectively (FIG.4e, FIG. 4f). The number of OVA-specific CD8+CD44hiCD62LloCD69hiTRM-like cells in the lungs was also 2.7-, 7.4, and 9.4-fold higher (P = 0.0316, P = 0.0021, and P = 0.0015, respectively) in the MDN mOVA LNP-immunized group in comparison to those immunized with PBT mOVA LNPs, DI-8 mOVA LNPs, and PBS, respectively (FIG. 4g). This demonstrates that MDN mOVA LNPs elicited stronger lung-specific CD8+ T cell immunity compared to the other LNP formulations.

[0213] To further assess the antigen- specific cytolytic functionality of the lymphocytes, the liver, lungs, and spleen of the vaccinated mice were collected on Day 28 and homogenized into a cell suspension for ex vivo antigen restimulation. Elevated production of pro-inflammatory cytokine after restimulation was detected across all three LNP formulations. Particularly, the PBT mOVA LNP-treated group showed a significantly greater number of IFN-y-secreting lymphocytes in the liver, with the frequency being 1.8- and 12.4-fold higher compared to the MDN mOVA LNP- and DI-8 mOVA LNP-treated groups, confirming the superior liver-biased immune response generation capability of the PBT mOVA LNPs (FIG. 4h, FIG. 31). In contrast, the MDN formulation induced the highest number of IFN-y-secreting lymphocytes in the lungs, reflecting its lung-bias characteristic (FIG. 4i, FIG. 32). In the spleen, the number of IFN-y-secreting lymphocytes was similar across all three LNP formulations, demonstrating comparable systemic cellular immune responses (FIG. 4j, FIG. 33). The frequency of OVA-specific CD8+T cells, specifically, in the spleen for the PBT mOVA LNP formulation, however, is 2.0- and 8.8-fold higher than that for the MDN and DI-8 LNP formulations, respectively, suggesting potential for more OVA-specific CD8+T cells in systemic circulation and subsequent homing to antigenexpressing tissues (FIG. 4k). In terms of humoral immunity, all three formulations elicited similar levels of OVA-specific IgG, IgGl, and IgG2c titers indicative of potent systemic humoral responses (FIG. 41-FIG. 4n). These results are consistent with the expectation that systemic5243985.601_P18418-02humoral immunity is primarily driven by local transgene transfection and CD4+helper T cell pathways, Swain et al., 2012, and less on the organ-specific trafficking of the LNPs.

[0214] Collectively, these data provide evidence that different LNP compositions can lead to marked differences in the magnitude and development of tissue- specific immunity within specific organs.

[0215] Assessment of long-term systemic and tissue-specific immune activation by selected mRNA LNP formulations following i.m. injection

[0216] Next, we assessed the longevity of the immune responses and memory formation generated by the selected LNP formulations to determine whether the local and systemic immunity observed at the earlier time point would be sustained over an extended period. To evaluate long-term immune responses, we followed the same prime-and-boost vaccination schedule with three i.m. injections of LNPs containing 10 pg of mOVA or PBS. On Day 90, the liver, lungs, spleen, and blood of the mice were collected for immune assessments (FIG. 5a). In line with the 28-day shortterm immune response data, the PBT LNP formulation continued to show a substantial retention of OVA-specific CD8+T cells (12.7- and 64.9-fold more than the MDN and DL8 LNP formulations, respectively) and OVA-specific CD8+CD44hiCD62LloCD69hiTRM-like cells (18.7-and 64.0-fold more than the MDN and DI-8 LNP formulations, respectively) in the liver. Notably, the number of these cells remained substantially higher than PBS baseline and the other treatment groups, with the difference becoming even more pronounced at this extended time point than the 28-day time point. As illustrated, the MDN and DI-8 LNP formulations showed minimal retention of OVA-specific CD8+T cells and TRM-like cells in the liver, showing levels close to the PBS baseline (FIG. 5b-FIG. 5d). In the lungs, a modest but discernible increase in the number of OVA-specific CD8+T cells (1.5- and 2.1-fold more than the PBT and DI-8 LNP formulations, respectively) and OVA-specific CD8+CD44hiCD62LloCD69hiTRM-like cells (1.6- and 3.4-fold more than the PBT and DI-8 LNP formulations, respectively) were observed in the MDN group at the 90-day mark, suggesting that MDN may favor the formation of longer-term immune memory in lung tissues (FIG. 5e-FIG. 5g). These frequencies, however, remained lower than those observed in the liver with the PBT formulation, highlighting the differences in targeting capability and room for further optimization for lung-targeting LNPs.

[0217] Interestingly, at the 90-day time point, in the spleen of mice vaccinated with the PBT LNPs, a significantly higher number of OVA-specific CD8+T cells (3.4- and 17.2-fold more than the5343985.601_P18418-02MDN and DI-8 LNPs, respectively) and CD8+CD44hiCD62LloCD69hiTRM-like cells (3.9- and 14.2-fold more than the MDN and DI-8 LNPs, respectively) was observed (FIG. 5h, FIG. 5i). To assess the antigen-specific cytolytic T cell responses, the spleen of the vaccinated mice were collected and homogenized into a cell suspension for ex vivo antigen restimulation. The three LNP-treated groups showed increased frequencies of CD3+CD8+TNF-a+cell population compared to the PBS group. In comparison to MDN and DI-8 LNPs, the PBT LNPs resulted in a significantly higher frequency of CD3+CD8+TNF-a+cells with P values of 0.0006 and <0.0001, respectively (FIG. 5j, FIG. 34). Additionally, the frequency of IFN-y-secreting lymphocytes in the spleen of PBT LNP-treated mice was 2.7- and 13.1-fold higher compared to MDN and DI-8 LNP-treated mice (FIG. 5k, FIG. 35). Without wishing to be bound to any one particular theory, it is thought that the biodistribution and transfection of mRNA LNPs in organs outside of the injection site may correlate with the ability of enhancing and maintaining systemic immunity.

[0218] Regarding humoral immunity, the anti-OVA IgG and IgGl titers detected at Day 90 were comparable across all three formulations, indicating that general antibody responses were relatively stable (FIG. 5l, FIG. 5m). The PBT LNPs, however, induced a significantly higher level of IgG2c titer than the MDN and DI-8 LNPs with P values of 0.0039 and 0.0032, respectively, pointing to a more potent Thl-biased immune response (FIG. 5n). This finding is consistent with our earlier data, which showed a sustained systemic effector T cell response, further reinforcing the role of the PBT LNP composition in promoting a durable Th 1 -driven immune response.

[0219] Taken together, these results provide evidence that the PBT LNP formulation not only supports robust immunity in the liver, but also enhances long-term systemic immune memory, particularly through retention of CD8+T cells in the spleen and promotion of a Thl-biased systemic immune profile. This finding indicates that LNPs differ substantially in their ability to induce both tissue- specific and systemic immune responses critical for long-lasting vaccine-mediated protection.

[0220] Therapeutic and prophylactic efficacy of mRNA LNP vaccine with liver-biased T cell recruitment after i.m. administration

[0221] Building on the localized antigen- specific CD8+T cell responses in the liver elicited by the PBT LNPs, we next evaluated its therapeutic potential as a cancer vaccine against liver tumor growth using OVA as the model antigen in mice. To address the challenge of accurately measuring tumor size within the liver, B16-OVA-fLuc cells, which are B16 melanoma cells genetically5443985.601_P18418-02engineered to constitutively express luciferase, were utilized to enable non-invasive monitoring of tumor growth via bioluminescence imaging. First, C57BL / 6 mice were inoculated with 5xl06B 16-OVA-fLuc cells via intrahepatic injection on Day 0. On Days 2, 4, and 6 post-tumor inoculation, the mice were immunized with PBT, MDN, or DI-8 LNPs containing 10 pg mOVA (FIG. 6a). Over the 30-day observation period, mice treated with PBT LNPs showed prolonged overall survival (FIG. 6b) and considerable tumor regression (FIG. 6c) in the B16-OVA-fLuc treatment model; their 30-day survival rate was 57% compared with median survival times of 6 days, 4 days, and 5 days for MDN LNP, DI-8 LNP, and PBS groups, respectively. In the PBT group, there was apparent tumor regression from Days 5 to 30 (FIG. 6d).

[0222] We next evaluated the efficacy of PBT LNPs as a prophylactic vaccine in the B16-OVA-fLuc mouse liver tumor model. C57BL / 6 mice were immunized on Days 0, 7, and 14 with 10 pg of PBT LNPs containing 10 pg mOVA. On Day 60, these mice were challenged with 5 x 106B 16-OVA-fLuc cells by intrahepatic injection (FIG. 6e). As illustrated in FIG. 6f, FIG. 6g, PBT-vaccinated mice had 100% survival rate during the 30-day observation period, compared to a median survival of 4 days for the PBS group. Although the PBT-vaccinated mice initially displayed a detectable increase in tumor burden, it was quickly controlled and reduced to nearbackground levels for all vaccinated mice. This highlights the ability of the PBT LNP formulation to elicit a protective immune response capable of controlling and eradicating tumor cells even after a delayed challenge.

[0223] Given the robust immune responses in the lungs induced by the MDN formulation, we further evaluated its antitumor efficacy as a therapeutic vaccine against metastatic lung tumors, providing additional evidence in a distinct organ that is highly susceptible to metastasis. C57BL / 6 mice were inoculated with 1 x 105B16-OVA cells via i.v. injection on Day 0. On Days 5, 8, and 11 post-tumor inoculation, mice were immunized with PBT, MDN, or DI-8 LNPs containing 10 pg mOVA (FIG. 36a). Body weight was monitored over time as an indicator of health status. While all tumor-bearing mice exhibited some degree of weight loss, MDN-treated mice experienced the least weight loss, which was significantly lower than in the PBS group (P = 0.0004) (FIG. 36b). On Day 21, lungs were harvested and examined for metastatic foci. The MDN group had the lowest average number of metastatic foci (7.57 ± 7.23). which was 71% lower than PBT (26.43 ± 17.85), 72% lower than DI-8 (27.11 ± 18.08). and 88% lower than PBS (62.50 ± 51.77) (FIG. 36c, FIG.37). These results confirm that the MDN formulation achieves potent antitumor therapeutic5543985.601_P18418-02efficacy by reducing metastatic tumor burden more effectively than the other tested formulations. This enhanced efficacy is consistent with MDN LNPs’ tissue-specific immune activation profile, which is characterized by strong lung-localized antigen expression and a high frequency of lungresident CD8+TRM-like cells, enabling rapid and targeted immune responses against metastatic tumor cells in the pulmonary microenvironment.

[0224] By directing LNPs to traffic and express the antigen in specific organs affected by metastatic tumors, it is possible to deliver immunotherapeutic agents directly to the tumor microenvironment. This tissue-targeted delivery and generation of TRM-like cells can enhance the local immune response against tumor cells, improving the efficacy of cancer immunotherapy.

[0225] Me chanistic insights into systemic mRNA LNP trafficking

[0226] To elucidate the key steps underlying systemic trafficking of mRNA LNPs, we employed a dual-component labeling strategy using PBT LNPs as a model formulation, given their highest trafficking capacity among the LNPs evaluated. In this system, lipid components were labeled with l,l'-dioctadecyl-3.3.3',3'-tetramethylindotricarbocyanine iodide (DiR), a near-infrared lipophilic dye, and the mRNA cargo was labeled with Cy5, enabling simultaneous tracking of both components. Additionally, luciferase-encoding mRNA was encapsulated to provide a functional readout of transgene expression. This integrated approach allowed comprehensive analysis of LNP trafficking and delivery dynamics.

[0227] C57BL / 6 mice were intramuscularly injected with labeled PBT LNPs encapsulating 20 pg of luciferase mRNA. DiR and Cy5 fluorescence signals, along with bioluminescence at the injection site and systemic organs, were monitored from 0 to 48 h post-injection. Both DiR and Cy5 signals at the injection site declined in parallel over time, with concurrent increases in systemic organs, indicating that LNPs predominantly trafficked as intact nanoparticles rather than disassembling locally (FIG. 7a, FIG. 7b). Interestingly, robust luciferase expression was detected in systemic organs at early time points, suggesting that distal transgene expression may contribute more significantly to vaccine efficacy than previously appreciated (FIG. 7c). This raises an important consideration that systemic expression could be a critical factor in mRNA vaccine design. Organ-level analysis revealed strong liver-dominant trafficking and transfection, confirming the liver tropism of PBT LNPs (FIG. 7d-FIG. 7df, FIG. 38-FIG. 39). To further confirm the transfection kinetics in the liver, ex vivo luciferase assay was performed on the cells isolated from the liver and illustrated a transfection trend that is similar to what was observed with the ex5643985.601_P18418-02vivo TVIS imaging, showing peak luminescence in the liver at 3 h post-LNP injection (FIG. 40). DiR and Cy5 signals exhibited similar kinetics across most organs, consistent with intact particle uptake, except in the kidneys, where a noticeable discrepancy between the two signals likely reflects partial mRNA release during trafficking and subsequent renal clearance (FIG. 7e). Wholebody imaging across the timepoints further supported this systemic trafficking pattern (FIG. 7g). Immediately after injection, both DiR and Cy5 signals were confined to the injection site, indicating that the LNPs remained localized within the muscle. Over time, both signals gradually dispersed away from the injection site. By approximately 3 hours post-injection, co-localized DiR and Cy5 signals became detectable in the liver, corroborating that most LNPs trafficked as intact nanoparticles rather than as disassembled components. In parallel, bioluminescence signals also spread from the injection site to the liver, demonstrating that mRNA LNP expression occurred in distal organs reached by the particles. This confirms that the trafficked LNPs not only arrived at those tissues but also retained their structural integrity, functionality, and transfection capability. Collectively, these findings indicate that the majority of particles trafficked intact in vivo and that the PBT formulation exhibited a pronounced liver-dominant trafficking and transfection profile.

[0228] We next examined the contribution of different potential pathways to mRNA LNP systemic trafficking, beginning with lymphatic drainage. Following i.m. injection of DiR- and Cy5-labeled mLuc LNPs, strong DiR and Cy5 signals were detected in the local inguinal lymph node, indicating that LNPs entered the local lymphatic system after i.m. injection (FIG. 7h, FIG. 7i). DiR and Cy5 fluorescence were also detected in the distal inguinal lymph node, although the signals were significantly lower than those in the local node, their presence suggests that the LNPs did access the broader lymphatic network and that lymphatic drainage may facilitate systemic trafficking (FIG. 7h, FIG. 7i). A minor degree of transfection was observed in the local inguinal lymph node, demonstrating that LNPs not only drained to the node but also transfected a small subset of cells there (FIG. 41). In the distal inguinal lymph node, bioluminescence levels were much lower than in the local node but increased over time before gradually declining, which indicates that LNPs reaching the distal lymph node mediated transient transfection there, as well (FIG. 41). These findings confirm that LNPs can access the lymphatic system and that lymphatic drainage contributes to systemic dissemination of the LNPs.

[0229] We then investigated the role of the bloodstream. Equal volumes of blood collected at various time points post-LNP injection were assessed via ex vivo IVIS imaging, which revealed5743985.601_P18418-02the presence of both DiR and Cy5 signals, indicating that LNPs circulate in the blood (FIG. 7j). To determine whether these circulating LNPs were cell-associated or free particles, circulating cells in the blood were isolated and analyzed by flow cytometry. Very low, near-background percentages of DiR+and Cy5+cells were detected, suggesting that the majority of LNPs in circulation were free particles rather than being internalized or carried by the circulating cells (FIG.7k— 1). Together, these results indicate that both lymphatic drainage and blood circulation contribute to the systemic trafficking of LNPs.

[0230] Compositional optimization of LNPs for enhanced liver-tropic trafficking and tissue resident immunity

[0231] To systematically correlate LNP composition with antigen expression and immune profiles, and to further optimize LNPs for enhanced liver-tropic trafficking and tissue-resident immunity, we constructed a comprehensive 649-member LNP library (FIG. 8a). All formulations used ALC-0315 as the ionizable lipid, DMG-PEG, cholesterol, and a helper lipid selected from those present in FDA-approved or experimental LNP formulations. Helper lipids were chosen to represent different charge characteristics, including cationic [l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyldioctadecyl ammonium (DDAB)], zwitterionic [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)], and anionic [1-stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (18PG) and bis(monooleoylglycero)phosphate (18BMP)]. Parameter spacings were based on our previous study: (1) ratio of ALC-0315 to helper lipid ranging from 1 to 200; (2) ratio of cholesterol to DMG-PEG2000 ranging from 10 to 500; (3) combined percentage of ALC-0315 and helper lipid ranging from 20% to 80%; and (4) N / P ratio ranging from 4 to 12. These parameter choices yielded a sufficiently diverse library, enabling us to confirm whether composition drives differences in trafficking behavior and to investigate how variations in antigen expression levels influence the tissue-specific immune responses observed with the initially tested formulations.

[0232] To identify LNP formulations with strong liver- specific transgene expression, we first evaluated the mRNA delivery efficiency of the library in HepG2 hepatocytes and C2C12 muscle cells using fLuc mRNA, quantifying the resulting luciferase expression. Top 50 composition candidates were selected based on high transfection efficiency in HepG2 cells while maintaining low transfection in C2C 12 cells (FIG. 8b). The in vivo transfection efficiency of LNPs likely differs from that of traditional in vitro assay screens due to the difference between in vitro and in vivo5843985.601_P18418-02settings and the associated delivery barriers, so the top candidates were next evaluated in vivo using a cluster-mode screening approach. The 50 formulations were organized into five clusters, with 10 formulations per cluster, according to helper lipid type. For each cluster, in vivo liver transfection efficiency was assessed following i.m. injection of fLuc mRNA at a dose of 5 pg per formulation (50 pg total per cluster). Although all selected LNPs demonstrated hepatocyte-biased transfection in vitro, in vivo performance varied substantially. Cluster 3 achieved the highest liver transfection efficiency, prompting further evaluation of its 10 individual formulations at 20 pg mRNA per mouse via i.m. injection (FIG. 8c). Even within the same cluster — sharing the same helper lipid but differing in composition ratios — performance ranged from superior to markedly inferior relative to the benchmark PBT LNP. LNP 10 exhibited the highest liver transfection, which was 181-fold greater than that of LNP 61 within the same cluster (FIG. 8d). In terms of in vivo biodistribution percentages, LNP 10 demonstrated a greater degree of systemic trafficking, with a higher transfection in major organs (44.21%) compared to PBT (36.28%), whereas LNP 61 showed a predominant transgene expression at the injection site and a minimal systemic transgene expression (11.29%) (FIG. 8e). Organ- specific analysis further underscored LNP 10’s advantage, achieving a higher percentage of luciferase expression in the liver (42.88%) than PBT (35.28%). Meanwhile, LNP 61 showed only 5% liver transfection, indicating limited transfection beyond the muscle injection site (FIG. 8f). To better quantify liver tropism of the evaluated LNP formulations, we calculated the liver-to-muscle luminescence ratio. LNP 10 exhibited the highest ratio, exceeding that of PBT and LNP 61 by 53% and 1,710%, respectively (FIG. 8g). We further validated the hepatocyte transfection efficiency of LNP 10 and LNP 61, representing the best and worst compositions within the cluster, using the Ai9 mouse model. On Day 7 after a single i.m. injection of 30 pg mCre LNPs, LNP 10 achieved the highest percentage of tdTom+cells in the liver (41.05%) compared to LNP 61 (0.24%, P = 0.0328) and PBS (0.08%, P = 0.0322) (FIG. 8h). Further characterization showed that these two compositions exhibited similar physical properties (e.g., particle size, surface charge, and encapsulation efficiency), all comparable to those of PBT LNPs (Table 1 and 2). This suggests that the observed differences in systemic trafficking and transfection profiles are likely attributable to changes in compositional features rather than to gross physicochemical differences. Based on these results, LNP 10 and LNP 61 were selected for downstream immune analyses as representatives of high and low liver-biased trafficking and transfection, respectively.5943985.601_P18418-02

[0233] We next investigated how biased LNP trafficking and organ-specific expression influence the generation of tissue- specific immunity. Short-term immune responses were evaluated following a prime-and-boost vaccination regimen using LNP 10 and LNP 61 encapsulating OVA as the model antigen. On Day 28, both formulations elicited comparable humoral immune responses, as indicated by similar anti-OVA IgG titers (FIG. 8i). However, in the liver, mice treated with LNP 10 exhibited 5.7-fold higher frequencies of OVA-specific CD8+T cells and 7.6-fold higher frequencies of OVA-specific CD8+CD44hlCD62LloCD69hiCD103+TRM cells compared to those treated with LNP 61 (FIG. 8j, FIG. 8k). These findings indicate that LNP 10 induced potent liver-specific cellular immunity and robust memory cell formation, highlighting that enhanced liver expression correlates positively with enhanced tissue- specific immunity without compromising systemic humoral responses. As LNP 10 also demonstrated enhanced transfection compared to PBT LNPs, we directly compared their ability to activate lymphocytes to further link transfection efficiency with immune activity. Following the same prime-and-boost vaccination schedule. LNP 10 induced a significantly higher frequency of IFNy- secreting lymphocytes in the liver on Day 28 than PBT LNPs (P = 0.0471) (FIG. 43 and FIG. 44). Collectively, these findings demonstrate that compositional differences can drive distinct systemic trafficking and transfection profiles following i.m. administration, and that variation in the extent of antigen expression within a specific tissue can substantially influence localized cellular immune responses and memory formation. These insights provide important guidance for the rational design of future mRNA vaccines.

[0234] Discussion

[0235] The systemic LNP trafficking behavior following i.m. and s.c. injections has been noted in previous literature. Pateev et al., 2024; Ma et al., 2024; Di et al., 2022; Hassett et al., 2024; Chen et al., 2022. These discussions, however, have been predominantly confined to the role of lymphatic drainage, with limited exploration into the concurrent entry of LNPs into the systemic circulation for therapeutic implications. Despite reports showing the trafficking of mRNA LNPs into other organs, attention has been on potential adverse effects due to the “leakage” rather than investigating their therapeutic potential. Pateev et al„ 2024; Ma et al., 2024; Di et al., 2022; Hassett et al., 2024. Furthermore, the relationship between LNP formulation composition and trafficking profiles, including distribution to and transgene expression in the liver and lungs following i.m.6043985.601_P18418-02injection, has remained largely unexplored, particularly regarding the associated potential therapeutic benefits.

[0236] One aspect of this Example lies in establishing a direct mechanistic link between LNP formulation-dependent trafficking patterns following i.m. administration, organ-specific transgene expression, and the induction of TRM cells, and in demonstrating how these insights can be exploited to rationally engineer LNPs that program immune responses toward specific tissues. By revealing that the site of antigen expression, not just total antigen expression, critically shapes tissue-specific T cell activation and memory formation, our study provides an engineering framework for intentionally directing antigen expression to desired tissues to enhance both local and systemic protection. This represents the first experimental demonstration that LNP trafficking from the i.m. injection site can be leveraged to tune TRM-mediated anti-tumor efficacy.

[0237] This Example explores the utility of systemic trafficking of the LNPs for therapeutic benefits. By comparing the biodistribution and transfection profiles of different LNP formulations, we identified those with distinct organ-biased expression properties, particularly the PBT LNPs showing a substantial liver-biased trafficking and antigen expression following i.m. injection. The PBT LNPs trafficked to and transfected the liver tissue, resulting in localized antigen expression, which, in turn, promoted the recruitment of antigen- specific T cells and enhanced TRM-like cell retention within the liver over 90 days. As a result, this LNP formulation demonstrated sustained local immunity and prolonged T cell effector function. Additionally, we observed that the humoral immune response was less dependent on systemic LNP trafficking. Despite differences in the extent of LNP trafficking from the injection site, the three selected LNP formulations examined in this study elicited comparable humoral immunity against the antigen of interest. This suggests that systemic LNP trafficking primarily influences T cell-mediated immunity, particularly in shaping antigen- specific CD8+T cell responses and CD8+TRM cell formation, while having minimal influence on mobilizing humoral immune responses. The dissociation between systemic LNP trafficking and humoral immunity highlights the distinct mechanisms governing B cell and T cell activation and recruitment in response to LNP-delivered antigens. These findings provide additional insights into how tissue-specific T-cell responses induced by LNP trafficking from the injection site can promote long-term immunity, with important implications for both localized and systemic defense mechanisms.6143985.601_P18418-02

[0238] Importantly, we also found that the magnitude of antigen expression is not linearly correlated with the magnitude of the immune response. For example, while circulating LNP levels after i.m. injection suggested roughly 48% relative bioavailability compared to i.v., direct measurements of absolute hepatic expression showed that i.v. dosing resulted in approximately 100-fold higher liver bioluminescence than i.m. injection. Yet, immune outcomes scaled differently: the 2 i.m. + 1 i.v. regimen yielded a ~6-fold increase in OVA-specific CD8+T cell responses compared to 3 i.m., underscoring that qualitative aspects of antigen expression and immune priming, rather than proportional increases in antigen load, dictate the ultimate immune outcome. This observation highlights the importance of both injection site priming and distal organ antigen expression in shaping tissue-specific immunity.

[0239] Previous research on LNPs has underscored the effect of LNP composition on tissuetargeting and transfection capabilities with i.v. injections. Safford et al., 2024; Lokugamage et al., 2021; Cheng et al., 2020; Oberli et al., 2017; Li et al., 2015; Zhu et al., 2024a; Xue et al., 2022; Zhu et al., 2022; Patel et al.. 2019; Zhu et al.. 2024b. Our findings expand on this by demonstrating that organ-specific LNP trafficking following i.m. injection can be similarly influenced by LNP composition. In this study, changing LNP formulations led to varying degrees of systemic trafficking and organ- specific transgene expression.

[0240] The ability to tailor LNP composition for enhanced and biased systemic trafficking may open new possibilities for creating LNPs with controllable trafficking profiles following i.m. injection for therapeutic purposes. One notable observation was that formulations with higher leakage capabilities also have higher PEGylate lipid content (Table 1 and 2). The hydrophilic nature of PEG contributes to reduced aggregation and longer circulation times, Suk et al., 2015, and possibly reduced binding with the extracellular matrix, which may allow for more efficient escape from the injection site and subsequent tissue distribution, offering a potential explanation for higher systemic trafficking out of the injection site. Further studies are needed to elucidate the exact mechanism by which LNP composition controls the systemic trafficking and tissue-specific antigen expression following i.m. injection, which could guide the development of more effective and targeted LNP-based therapeutic vaccines.

[0241] Tissue-specific CD8+T cells, particularly CD8+TRM cells, have gained attention for their role in long-lasting protection in tissues as entry points for pathogens. Fernandez-Ruiz et al., 2016; Mueller and Mackay, 2016; Okla et al., 2021; Nizard et al., 2017. These cells are recruited6243985.601_P18418-02following antigen expression in local tissues, with some establishing long-term residence to deliver localized, rapid immune responses upon antigen re-exposure. Park and Kupper, 2015; Mueller and Mackay, 2016; Dijkgraaf et al.. 2019; Ariotti et al., 2012; Schenkel and Masopust, 2014; Jiang, et al., 2012; Klonowski et al., 2004; Sallusto et al., 1999.

[0242] In various infectious disease studies, TRM cells have been shown to play a critical role in protection against infection, highlighting their potential in therapeutic strategies. Fernandez-Ruiz et al., 2016; Nizard et al., 2017; Ariotti et al., 2012; Jiang et al., 2012; Gebhardt et al., 2009; Pandey et al., 2024. By inducing tissue-specific T cell immunity beyond systemic immune responses, robust and durable protection can be conferred in tissues where pathogens may enter and / or reside, offering a promising dual-protective strategy in vaccine development. Our study illustrates the utility of this approach in preventing tumor recurrence, leveraging both systemic and local immune responses to enhance overall efficacy.

[0243] In this proof-of-concept mouse liver tumor study, we identified that the PBT LNP formulation, with more efficient trafficking to and transgene expression in the liver following i.m. injection, exhibited the strongest capacity for controlling and suppressing liver tumor engraftment, extending survival in both therapeutic and prophylactic models. This LNP formulation effectively mobilized immune defenses to prevent tumor recurrence by engaging tissue-specific CD8+T cells including TRM cells while sustaining systemic immunity. This approach is particularly valuable for preventing tumor recurrence after surgical excision by maintaining immune surveillance in the tissue where the tumor initially resided. In the lung metastasis model, the MDN formulation, which drives strong lung-localized antigen expression and TRM-like cell formation, markedly reduced metastatic burden, offering a strategy for treating metastatic tumors at common secondary sites such as the liver and lungs. This approach may help prevent tumor spread to critical organs, thereby reducing the risk of secondary tumor formation. Furthermore, the principles demonstrated in this tumor model could be applied to diseases like malaria, where targeting the liver during the latency period in hepatocytes could prevent disease progression. This highlights the broader potential of LNP-based vaccines to address both infectious diseases and cancers by tailoring immune responses to specific tissues.

[0244] Additionally, it may help prevent tumor metastasis to critical organs, reducing the risk of secondary tumor formation. From a tumor immunotherapy perspective, modulating LNP trafficking to target tumor-residing tissues offers a more effective strategy for tumor control and6343985.601_P18418-02clearance. Furthermore, the principles demonstrated in this tumor model could be applied to diseases like malaria, where targeting the liver during the latency period in hepatocytes could prevent disease progression. This highlights the broader potential of LNP-based vaccines to address both infectious diseases and cancers by tailoring immune responses to specific tissues.

[0245] In summary, this Example provides insights into systemic trafficking of LNPs from the injection site following i.m. injection and revealed that mRNA LNP compositions exhibiting a higher propensity for systemic entry following i.m. injection and subsequent antigen expression in selected organs, induced localized antigen-specific CD8+T cell response and the formation of TRM-like cells. Using a tumor vaccine as a proof-of-concept case study, we showed that the PBT mRNA LNPs were capable of generating antigen expression and T cell recruitment to the liver beyond the systemic immunity following i.m. immunization, resulting in enhanced antitumor efficacy in both therapeutic and prophylactic liver tumor models. These findings have important implications for the design of targeted vaccines and therapeutics where precise tissue- specific and durable immunity are key. The ability to finetune LNP composition to achieve desired leakage profiles through i.m. injection not only enhances therapeutic efficacy, but also opens new possibilities for creating highly specific and effective treatments for a range of diseases.

[0246] Methods

[0247] Materials

[0248] DLin-MC3-DMA was obtained from MedKoo Biosciences, and SM-102 and ALC-0315 were from Broadpharm. DSPC, DOPE, 18PG, DOTAP, DDAB, 18BMP, and DMG-PEG-2000 were from Avanti Polar Lipids, and cholesterol was from Sigma- Aldrich. The B16-OVA-fLuc cells, a luciferase reporter cell line, were obtained from AcceGen. HepG2 cells and C2C12 cells were purchased from American Type Culture Collection, USA (ATCC). B16-OVA cells (expressing model antigen, OVA, with a transmembrane domain) were kindly provided by the lab of Prof. Jonathan Schneck. Reporter lysis buffer and luciferin assay solution were purchased from Promega. D-Luciferin (sodium salt) was acquired from Gold Biotechnology. All mRNA constructs, except for the Cy5-labeled mRNAs, were purchased from TriLink BioTechnologies, capped using their CleanCap proprietary co-transcriptional capping method, and designed form the naturally occurring Cap 1 structure with a high efficiency. The mRNAs were also polyadenylated, modified with 5-methoxyuridines, and optimized for mammalian systems. Cy5-labeled fLuc mRNA was obtained from ApexBio. 1,1 '-dioctadecyl-3,3,3',3'-6443985.601_P18418-02tetramethylindotricarbocyanine iodide (DiR) was purchased from Invitrogen.Table 4. Chemical structures of ionizable lipids in the evaluated LNP formulations.Ionizable Lipid Chemical StructureL 6ALC-0315aDlin-MC3-DMAL G SM-102

[0249] LNP synthesis

[0250] LNPs were made by mixing an organic phase consisting of the lipid components and an aqueous phase consisting of the gene cargo. The organic phase was prepared by dissolving a mixture of ionizable lipid (ALC-0315, SM-102, or DLin-MC3 DMA), cholesterol, DMG-PEG2000, and a helper lipid selected from a group consisting of DDAB, DOTAP, DSPC. DOPE, 18PG, or 18BMP, at predetermined ratios in ethanol. To prepare DiR-labeled PBT LNPs, DiR was added to the lipids in the ethanol at a molar ratio of 0.25%. The mRNA cargo (OVA mRNA, fLuc mRNA, Cre mRNA, or Cy5-labeled fLuc mRNA) was dissolved in 25 mM magnesium acetate buffer at a pH of 4.0. All mRNA samples were stored at -80 °C and thawed on ice before use. The flash nanocomplexation (FNC) device was used to mix the ethanol and aqueous phases at a 3:1 ratio using syringe pumps using a previously reported protocol49. Then the LNPs were purified through dialysis against DI water with a 100-kDa MWCO cassette at 4°C for 24 h and stored at 4°C before injection. For high-throughput screening, the MANTIS liquid handler (Formulatrix) was used to prepare the 649-member LNP library in 96-well plates according to an input file specifying formulation compositions.

[0251] LNP characterization6543985.601_P18418-02

[0252] The zeta potential, average size, and polydispersity index (PDI) of LNPs were measured using dynamic light scattering (ZetaPALS, Brookhaven Instruments) method, with diameters reported as the intensity average.

[0253] A Quant-iT RiboGreen assay (ThermoFisher, R11490) was utilized to quantify mRNA concentrations and assess the encapsulation efficiency (EE) of LNP formulations. Standard curves were prepared in duplicate on a black 96-well plate with mRNA concentrations ranging from 0.1 to 1.0 pg / mL in each well. Samples of LNPs were added in triplicate at a consistent LNP to DI water ratio of 1:20. Following this, 60 pL of RiboGreen solution — a 200-fold dilution of RiboGreen dye in water — was added to each well. Fluorescence measurements were taken using a BioTek Synergy Hl plate reader (excitation: 480 nm, emission: 520 nm) to determine mRNA levels in intact LNPs. Following the initial reading, 10% w / v Triton-X was added to each well to disrupt the LNP structure and release the encapsulated mRNA. Similar to the first reading, 60 pL of RiboGreen solution was added to the wells. Fluorescence readings were measured to determine the total mRNA content post-release. The measured fluorescence signals before and after mRNA release were converted to mRNA concentration using the standard curves. The encapsulation efficiency (EE%) of the LNPs were calculated using the following equation.

[0254] Total mRNA Concentration

[0255] Cell culture and high-throughput screening for transfection studies

[0256] For monolayer culture studies, HepG2 cells (ATCC) or C2C12 cells (ATCC) were seeded into 96-well plates at a cell density of 10,000 cells per well one day prior to transfection. LNPs were pipetted into cell medium at a final concentration of 1 pg / mL of mRNA. For example, 5 pL of LNPs suspension at 20 pg / mL of mRNA was pipetted into the 100 pL culture medium in each well. The transgene expression was analyzed following 24 h incubation. When characterizing luciferase as the reporter, cells were lysed by reporter lysis buffer (Promega) using two freezethaw cycles, and luciferase expression of the lysate was analyzed using a luminometer following a standard protocol using the luciferin assay solution (Promega).

[0257] Animals and primary cells

[0258] All animal procedures were conducted in accordance with protocols approved by the Johns Hopkins Institutional Animal Care and Use Committee (Protocol #MO24E165). C57BL / 6 mice (male and female), aged 6-8 weeks, were obtained from the Jackson Laboratory. Ai9 mice (male and female), 6-8 weeks old, were bred in the Johns Hopkins Animal Facilities and assigned6643985.601_P18418-02randomly to groups in the studies. Similarly, male and female OT-I mice, aged 6-8 weeks, were bred in the same facilities and randomly grouped. The mice had free access to pelleted feed and water, with the feed typically containing 5% fiber, 20% protein, and 5-10% fat. On average, the mice consumed 4-5 g of pelleted feed (120 g per kg body weight) and drank 3-5 mL of water (150 mL per kg body weight) daily. The temperature in the mouse rooms was maintained between 18-26 °C (64-79 °F) with 30-70% relative humidity, ensuring at least 10 air changes per hour. The mice were housed in standard shoebox cages with corncob bedding.

[0259] The LNPs were given through i.m. (right quadriceps) injection or i.v. (lateral tail vein) injection at a predetermined dose per mouse. The LNP suspensions were concentrated to 200 pg / mL for i.m. injection or 100 pg / mL for i.v. injection of mRNA by an Amicon Ultra-2 centrifugal filter unit with an MWCO of 100 kDa. The D-luciferin solution was given through i.p. injection (lower quadrant of abdomen) at a predetermined dose per mouse. For experiments in Ai9 mice, the Cre mRNA LNP formulations were prepared as described above and administered via i.m. or i.v. injections at an mRNA dose of 10 pg per mouse. For experiments described in FIG.20-19, the LNP suspensions were concentrated to 100 pg / mL and 200 pg / mLfor the 100 pL and 50 pL injection groups.

[0260] For the OT-I cell migration experiments, primary CD8 T cells were obtained from homogenized spleens and inguinal lymph nodes of OT-I mice using a CD8+T cell isolation kit and LS columns (Miltenyi Biotec), following the manufacturer’s protocols. Briefly, splenocytes were obtained from the homogenized suspension by lysing red blood cells with the ACK lysis buffer (ThermoFisher Scientific) and incubated with the supplied antibody cocktail and magnetic beads, then passed through a magnetic column to obtain a purified population. Isolated CD8+T cells were stained with CellTrace Violet cell proliferation kit (ThermoFisher, C34571). Cell counts were adjusted to 2.25 x 106per 100 pL in PBS, and mice were injected i.v. (lateral tail vein) with 100 pL of cell suspension.

[0261] Tissue processing and cell isolation

[0262] For isolation of cells from the liver, lungs, and spleen in the Ai9 mouse experiments, the harvested tissues were placed onto 40-pm cell strainers and digested mechanically with the back of a 3-mL syringe plunger in PBS. The cells were pelleted at 300 xg for 5 min at 4 °C, followed by resuspension in the ACK lysis buffer and incubation at room temperature for 7 min to lyse red blood cells. Cells were then pelleted by centrifugation at 300 xg for 5 min at 4 °C, washed with6743985.601_P18418-02PBS and pelleted twice before staining for flow cytometry. All steps were performed protected from light.

[0263] For isolation of lymphocytes from the liver, harvested livers were placed onto 40-pm cell strainers. The livers were gently dissociated by pressing through the strainer with the back of a 3-mL syringe plunger in circular or back-and-forth motions, and the resulting cell suspensions were collected in sterile 50-mL tubes. The cell strainer and plunger were rinsed with 15 mL of RPMI-1640 media, followed by an additional 15-mL rinse, for a total of 30 mL RPMI-1640 media. The tubes containing the liver cell suspension were swirled to ensure even distribution and kept on ice. To pellet gross hepatocytes, tubes were centrifuged at 77 xg for 1 min at 4 °C without brake. The supernatant containing lymphocytes was transferred to fresh 50-mL tubes, and the volume was adjusted to 50 mL with RPMI-1640 media. Cells were pelleted by centrifugation at 478 xg for 8 min at 4 °C with brake. After aspirating the supernatants, the cell pellets in each tube were resuspended in 10 mL of the 35% Percoll solution (Millipore-Sigma), prepared in HBSS / Heparin solution. Cell suspensions were then centrifuged at 850 xg for 25 min at room temperature without brake. Following centrifugation, the top layer containing hepatocytes and most of the Percoll solution was carefully aspirated. The lymphocyte pellets were resuspended in 2 mL of ACK lysis buffer and incubated at room temperature for 5 min to lyse red blood cells. Eight mL of MACS buffer was then added, and the cells were pelleted by centrifugation at 417 xg for 8 min at 4 °C with brake. Isolated lymphocytes were suspended in RPMI-1640 media for subsequent analyses.

[0264] For isolation of lymphocytes from the spleen, harvested spleens were placed onto 40-pm cell strainers. The spleens were mechanically digested through the cell strainers with the back of a 3-mL syringe plunger in a lymphocyte separation medium (PromoCell). The resulting single-cell cell suspensions were transferred to sterile 15-mL tubes, and 1 mL of RPMI-1640 media was slowly added to each tube to form visible layers. The tubes were then centrifuged at 800 xg for 25 min at 4 °C without brake. Following centrifugation, the middle layer containing lymphocytes was carefully collected and transferred to fresh 15-mL tubes, and 5 mL of RPMI-1640 media was added and mixed thoroughly. The cells were pelleted by centrifugation at 300 xg for 5 min at 4 °C. The pellet was then resuspended in 3 mL of RPMI-1640 media and centrifuged again at 300 xg for 5 min at 4 °C. Isolated lymphocytes were suspended in RPMI-1640 media for subsequent analyses.

[0265] For isolation of lymphocytes from the lung, harvested lungs were first minced into smaller pieces using surgical scissors and transferred to sterile 50-mL tubes. Lung digestion solution was6843985.601_P18418-02prepared with RPMI-1640 and collagenase type 1 (45 U / pL collagenase I). Ten mL of lung digestion solution was added to each tube containing the minced lung tissue, and the samples were incubated on a shaker at 37 °C for 1 h. The digested samples were then further processed mechanically with the back of a 3-mL syringe plunger through 40-pm cell strainers. RPMI-1640 medium was used to wash cells through the filters into sterile 50-mL tubes. The cell suspensions were then centrifuged at 500 xg for 5 min at 4 °C. After centrifugation, the cell pellets were resuspended in 2 mL of ACK lysis buffer and incubated at room temperature for 5 min to lyse red blood cells. Eight mL of MACS buffer was then added, and the cells were pelleted by centrifugation at 500 xg for 5 min at 4 °C with brake. Isolated lymphocytes were suspended in RPMI-1640 media for subsequent analyses.

[0266] Antibodies and staining for flow cytometry

[0267] Antibody panels are provided in Table 3. All antibodies were diluted at a ratio of 1:100 before use. LIVE / DEAD fixable dead cell stain kits were used to determine the viability of cells. CellTrace Violet cell proliferation kit (ThermoFisher, C34571) was used to stain and monitor OT-I cells in vivo. eBioscience Foxp3 / Transcription Factor Staining buffer set (ThermoFisher, 00-5523-00) was used for intracellular staining.

[0268] Table 3. Anti-mouse antibodies used in flow cytometry panels. Marker, fluorophore, catalogue number, source, and concentration are indicated.CatalogueAntigen Fluorophore Source Concentration #CD8a APC-Cy7 100714 Biolegend 1:200 dilution OVA SIIGFEKL NIH tetramerBrilliant Violet 421 N / A 1:400 dilution (Tetramer) core facilityCD3 APC 100236 Biolegend 1:100 dilution Thermo 1:1000 Live / Dead Live / Dead Fix Aqua L34957Fisher dilution CD44 FITC 103006 Biolegend 1:100 dilution BD CD44 PerCP-Cy5.5 560570 1:100 dilution BiosciencesCD62L PE 161204 Biolegend 1:100 dilution6943985.601_P18418-02CD62E Brilliant Violet 650 104453 Biolegend 1:100 dilution CD45 FITC 103108 Biolegend 1:250 dilution CD31 APC-Cy7 102440 Biolegend 1: 100 dilution CD326 Brilliant Violet 605 118227 Biolegend 1:100 dilution CD45 Brilliant Violet 421 103134 Biolegend 1:250 dilution CD3 PE 100206 Biolegend 1:200 dilution CD8 FITC 100706 Biolegend 1:200 dilution CD8a Brilliant Violet 750 747134 Biolegend 1:200 dilution CD45R PE-Cy7 103222 Biolegend 1:100 dilution CD69 Alexa Fluor 700 104539 Biolegend 1:50 dilution CD69 FITC 104506 Biolegend 1:50 dilution TNF alpha Alexa Fluor 700 506338 Biolegend 1:100 dilution CD3 Brilliant Violet 421 100228 Biolegend 1:200 dilution CD326 APC 118214 Biolegend 1:100 dilution CD 103 APC 121414 Biolegend 1:100 dilution

[0269] Isolated cells from the tissues, as described in the previous section, were resuspended and pelleted in 100 µL of antibodies diluted in flow cytometry staining buffer obtained from eBioscience™. The cells were then incubated on ice in the dark for 1 h. After the incubation period, the stained cells were washed twice with PBS and subsequently resuspended in 200 µL of eBioscience™ flow cytometry staining buffer for flow cytometry analysis. Flow data was acquired using an Attune NXT flow cytometer and analyzed with FlowJo software v.10.

[0270] Enzyme-linked immunosorbent spot (ELISpot) assay and FluoroSpot assay

[0271] For the ELISpot assay, multiscreen filter plates (Millipore-Sigma. S2EM004M99) were coated with antibodies targeting IFN-y (BD Biosciences, 551881) and subsequently blocked according to the manufacturer’s instructions. A total of IxlO6isolated lymphocytes were then added to each well and stimulated with SIINFEKL peptide at a concentration of 2 pg / mE for 18 h. The spots were detected using a mouse IFN-y detection antibody (BD Biosciences, 551881),7043985.601_P18418-02followed by an incubation with streptavidin-HRP (BD Biosciences, 557630) and AEC substrate (BD Biosciences, 551951). The plates were then forwarded to the SKCCC Immune Monitoring Core for further analysis. For the FluoroSpot assays, the Mouse IFN y FluoroSpot Plus kit (Mabtech) was utilized, adhering to the manufacturer’s protocols. In this case, 5×105isolated lymphocytes were plated per well and stimulated with SIINFEKL peptide at 2 pg / mL for 18 h before sending the plates to the SKCCC Immune Monitoring Core for evaluation.

[0272] Enzyme-linked immunosorbent assay (ELISA)

[0273] For serum antibody detection, 100 pF of blood sample was drawn from the tail vein of immunized mice on the day of sacrifice. Levels of antigen- specific IgG, IgGl, and IgG2c in the serum were measured by ELISA. Flat-bottomed 96-well plates (Nunc) were precoated with OVA protein at a concentration of 2 pg protein per well in 100 mM carbonate buffer (pH 9.6) at 4°C overnight, which were then blocked at room temperature for 2 h with blocking buffer, which consisted of 10% BSA in PBS. The serum obtained from mice was first diluted 30 times in the blocking buffer, followed by threefold serial dilutions. After blocking was done, the plates were washed with PBS-T (PBS containing 0.05% Tween), and the diluted serum samples were then added to the wells and incubated at 4 °C overnight. Horseradish peroxidase-conjugated goat antimouse IgG, IgGl, and IgG2c (Southern Biotech Associates) were used at a dilution of 1:2,000, 1:4,000, and 1:4,000, respectively, in the blocking buffer for labeling. After 1-h antibody incubation at room temperature, the plates were washed with PBS-T, followed by incubation with TMB ELISA substrate solution at room temperature. After a 30-min incubation, 50 pL of 4 N sulfuric acid was added to the wells to quench the reaction. The plates were then read at a wavelength of 450 nm with a plate reader. A sample was considered positive if its absorbance was twice as much as or higher than the absorbance of the negative control.

[0274] Immunization and tumor experiments

[0275] For immunization, C57BL / 6 mice aged 6-8 weeks were i.m. injected in their right quadriceps with different LNPs containing 10 pg of OVA mRNA, as described in the main text. For all vaccination studies, a total of three doses were given. In prophylactic and therapeutic liver tumor studies, mice were injected via intrahepatic injection with 5 x 106B16-OVA-fLuc cells. In therapeutic studies, vaccinations began on Day 2 after tumor inoculation. Tumor growth was measured three times a week through whole-body imaging using IVIS. The D-luciferin solution was given through i.p. injection (lower quadrant of abdomen) at a predetermined dose per mouse.7143985.601_P18418-02Mice were euthanized when the total luminescence flux from the tumor exceeded 8 x 107p / sec / cm2 / sr.

[0276] In the metastatic lung tumor model, mice were injected via intravenous injection with 1×105B16-OVA cells. Vaccinations began on Day 5 after tumor inoculation to permit tumor establishment in the lungs. Body weight was measured every three days starting from the first vaccination. Mice were monitored daily for signs of distress and were euthanized if they experienced >20% loss of initial body weight or exhibited clinical signs of distress, in accordance with lACUC-approved endpoints.

[0277] Statistics

[0278] Unpaired t-tests were performed when comparing two groups. One-way analysis of variance (ANOVA) and Tukey’s multiple comparisons were performed when comparing more than two groups. Two-way ANOVA and Tukey’s multiple comparisons were used for comparisons of time-course data (e.g. body weight over time) across groups. Survival curves were compared using log-rank Mantel-Cox test. Statistical analysis was performed using Microsoft Excel and Prism 10 (GraphPad). A difference was considered significant if P < 0.05 (*P < 0.05, **P <0.01, ***P <0.001, ****P <0.0001).EXAMPLE 2

[0279] Lipid Nanoparticle Formulations Capable of Migrating to Systemic Organs Following Intramuscular Administration

[0280] Background

[0281] Lipid nanoparticles (LNPs) have been designed as an immuno stimulatory delivery platform for antigen-encoding mRNA in various prophylactic and therapeutic applications. The clinical success of LNPs in the recent development, for example, of COVID- 19 mRNA vaccines, has underscored their potential to revolutionize vaccine technology, providing a robust and versatile platform for rapid and effective vaccine development.

[0282] Intramuscular (i.m.) injection is the most widely employed route for administering vaccines, as it is relatively straightforward to administer and generally well-tolerated for repeated dosing. Despite the proven efficacy of i.m. delivery of LNP vaccines, there is a significant gap in understanding the post-i.m. injection behavior of LNPs, particularly concerning their potential leakage from the injection site and the related opportunities and applications. Leakage refers to the7243985.601_P18418-02migration of LNPs from the injection site into surrounding tissues or the bloodstream, in turn reaching other organs in the body. This phenomenon has profound implications for the biodistribution and efficacy of LNP-based vaccines. The extent of LNP leakage following i.m. injection could influence the overall distribution of the vaccine within the body, potentially mobilizing different immune cell populations and enhancing the desired immune response. This characteristic, in turn, unveils novel avenues for vaccine delivery, where controlled leakage could be harnessed to target specific tissues or organs, thereby enhancing the versatility and precision of LNP-based vaccines.

[0283] Overview

[0284] Such a leakage process allows for access of systemic organs through alternative pathways that will reduce non-specific delivery through unique biodistribution profiles. For example, controlled leakage of LNPs carrying mRNAs encoding a malaria antigen could be designed to reach the liver, where the malaria parasite first infects and matures. By targeting the liver, the vaccine could induce a more effective immune response at the initial site of infection, potentially providing better protection against the parasite. Another example could involve the development of an LNP-based human immunodeficiency virus (HIV) vaccine that utilizes leakage to target mucosal tissues, where HIV exposure often occurs, thereby providing a more robust and localized immune defense at the site of potential infection.

[0285] Recent studies have shown that the composition of LNPs could be engineered to alter the biodistribution and transfection behavior of the LNPs, achieving tissue-and cell-specific transgene expression for more controlled immune modulation. The detailed characteristics of LNPs that are capable of efficient leakage and the mechanism of leakage, however, are not clear. In this Example, we employed a multi-step screening platform to identify key LNP component features that enable liver-targeted leakage following i.m. injections. We also investigated and elucidated potential immunomodulatory pathways and pinpointed promising medical applications associated with this tunable leakage behavior of LNPs.

[0286] Results

[0287] mRNA LNP library design for multi-step screening

[0288] The entire LNP library shared ALC-0315 as the ionizable lipid and DMG-PEG2000 as the PEGylated lipid. Helper phospholipid choice was incorporated into this library, encompassing a total of six helper phospholipids that were previously used in experimental or FDA-approved LNP7343985.601_P18418-02formulations to represent a diverse array of phospholipid charges and structures: the anionic bis(monooleoylglycero)-phosphate (18BMP) and l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (18PG); the zwitterionic l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and DSPC; and the cationic l,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyl dioctadecyl ammonium (DDAB). Using ALC-0315, cholesterol, DMG-PEG2000, and one of the six helper lipids, we designed an initial library of 649 LNP formulations by varying the following parameters: (1) ratio of ALC-0315 to helper lipid ranging from 1 to 200; (2) ratio of cholesterol to DMG-PEG2000 ranging from 10 to 500; (3) combined percentage of ALC-0315 and helper lipid ranging from 20% to 80%; and (4) N / P ratio ranging from 4 to 12. These parameter choices provided us with a sufficiently diverse library of LNP formulations, with which we programmatically assessed LNP-mediated mRNA delivery following i.m. injections.

[0289] In vitro high-throughput screening identified top mRNA LNP formulations with high liver cell transfection and low muscle cell transfection

[0290] To select LNP formulations with strong liver- specific but minimal muscle-specific transgene expression, we first evaluated the mRNA delivery efficiency of the entire library using firefly luciferase mRNA. We measured luciferase protein expression in HepG2 cells (a human liver cancer cell line) and C2C12 cells (a mouse myoblast cell line). By varying the helper lipid choice and the aforementioned parameter ratios in the LNP compositions, transgene expression levels varied from formulation to formulation (FIG. 36). Of the 649 mRNA LNP formulations, 50 formulations demonstrated strong transfection in HepG2 cells accompanied by weak transfection in C2C12 cells in vitro.[hh2L\\Cluster-niode in vivo screening pinpointed the top LNP cluster with the highest liverspecific mRNA delivery following i.m. administration

[0292] We next assessed the in vivo delivery efficacy of the top 50 LNP compositions in mice following one i.m. injection. In the initial in vivo screening step, we adopted a cluster-mode screening approach, which grouped the top 50 LNP formulations into five clusters based on shared properties, each encompassing ten LNP compositions in total. These clusters underwent initial screening via i.m. injection to evaluate transgene expression levels in the injection site and major organs including liver, lungs, spleen, kidneys, and heart. The leakage behavior and tissue-specific delivery of each cluster were examined by delivering luciferase-encoding mRNA at a total dose of 50 pg mRNA per mouse (5 pg mRNA per formulation and ten formulations per mouse) via i.m.7443985.601_P18418-02injection. As shown in FIG. 37, different LNP clusters exhibited varied leakage from the injection site to the major organs and differential transfection behavior following the leakage. While Clusters 4-5 had high percentages of LNP accumulation and transfection in the muscle (74.58% and 89.27%), Clusters 1-3 showed a stronger tendency of leakage from the injection site (65.54%, 64.38%, and 70.93% transfection in the muscle) and remarkably higher transgene expressions in the liver. Within Clusters 1-3, different off-target effects (transgene expression in organs other than the liver) were observed, with Cluster 3 demonstrating the highest total luminescence flux in the liver and minimal off-target leakage into the other organs (0.43%) (FIG. 37B, FIG. 37C). The ten LNP formulations within Cluster 3, which all had DOPE as the helper lipid, were hence selected to be evaluated individually in the subsequent in vivo assessment.

[0293] Individual in vivo assessment of the ten formulations in the top cluster demonstrated distinct leakage behavior of different LNP compositions and identified LNP compositions with high liver preferentially following leakage

[0294] The transfection efficiencies of the ten individual LNP formulations within Cluster 3 (Compositions 10, 41, 42, 49, 61, 67, 68, 70, 71, 72) and Composition 649 (a formulation that has shown considerably high leakage percentage and liver preferentially) were further examined following i.m. injection at an mRNA dose of 5 pg per mouse using the same fLuc mRNA payload. Within Cluster 3, distinct leakage transfection behaviors were observed in the individual LNP formulations. Certain formulations (Compositions 10, 49, 67, 68, and 649) achieved markedly higher liver preferentially, with more than 80% of the transgene expression localized in the liver following leakage, compared to the other formulations within the same cluster (FIG. 38A, FIG.38B). The same five formulations (Compositions 10, 49, 67, 68, and 649) also exhibited the highest total bioluminescence flux in the liver following i.m. injection (FIG. 38C).

[0295] Comparative analysis of the top LNP compositions offered insights into key LNP features potentially governing the leakage effects of LNPs following i.m. injections

[0296] To extract the effect of formulation parameters on liver transfection following i.m. injection, a detailed comparative analysis was conducted on the in vivo transfection efficiency results of the set of ten LNP formulations in Cluster 3 and LNP Composition 649. As FIG. 39 shows, LNP formulations with relatively high liver- specific transfection efficiencies were those with (1) low ionizable lipid ALC-0315 molar percentages in a range of 10% to 60%; (2) low helper lipid molar percentages in a range of 0% to 40%; (3) high cholesterol molar percentages in a range7543985.601_P18418-02of 40% to 90%; (4) moderate PEGylated lipid molar percentages in a range of 0.1% to 5%; and (5) moderate N: P ratios in a range of 4 to 15. These LNP compositional features are identified to enable more efficient LNP leakage from the injection site to the organs and highly liver- selective transgene expression following the leakage.

[0297] Utilities

[0298] Prophylactic vaccines

[0299] One promising application of organ-targeted LNP leakage following i.m. injections is in the development of vaccines for diseases that tend to localize in specific tissues. For example, respiratory diseases such as influenza and COVID-19 primarily affect the lungs. By directing LNPs to accumulate in the lungs, vaccines can stimulate a stronger local immune response, enhancing protection against respiratory pathogens. Similarly, for HIV, which targets immune cells in the lymphoid tissues, organ-targeted LNPs can improve the delivery of antigens to these critical sites, potentially increasing the efficacy of the vaccine. Malaria, caused by parasites that primarily infect the liver before spreading to red blood cells, can benefit from LNPs designed to preferentially target and accumulate in the liver. This targeted approach can enhance the immune system's ability to recognize and attack the parasites early in their lifecycle, providing better protection against malaria. Additionally, diseases like tuberculosis, which affect the lungs, and hepatitis B and C, which target the liver, can also benefit from organ-targeted LNP vaccines. By focusing the immune response on the primary site of infection, these vaccines can offer more effective and long-lasting protection. Organ-targeted LNP leakage following i.m. injection can thus transform the landscape of prophylactic vaccine development, offering a more efficient and precise method to combat diseases that predominantly affect specific organs or tissues.

[0300] Organ-targeted gene editing

[0301] Intramuscular injection of LNPs can also be harnessed for organ-targeted gene editing. By engineering LNPs to selectively leak into specific organs, such as the liver or the lungs, it is possible to deliver gene editing tools like CRISPR-Cas9 directly to the target site. This approach could revolutionize the treatment of genetic disorders, allowing for precise and efficient editing of disease-causing genes in situ. The controlled leakage of LNPs could enhance the specificity and reduce the off-target effects of gene editing therapies, improving their safety and efficacy.

[0302] Organ-specific cancer immunotherapy7643985.601_P18418-02

[0303] Another potential application of organ-targeted LNP leakage is in the field of tumor immunotherapy. By directing LNPs to accumulate in specific organs affected by metastatic tumors, it is possible to deliver immunotherapeutic agents directly to the tumor microenvironment. This targeted delivery can enhance the local immune response against tumor cells, improving the efficacy of cancer immunotherapy. Organ-specific accumulation of LNPs could also reduce systemic toxicity and side effects, making cancer treatments more tolerable for patients. Beyond the aforementioned therapeutic application, organ-targeted LNPs can also be administered as a prophylactic vaccine post tumor excision to prevent remaining tumor cells from metastasizing to major organs.REFERENCES

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[0355] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.8143985.601_P18418-02

Claims

THAT WHICH IS CLAIMED:

1. A composition comprising a lipid nanoparticle comprising an ionizable lipid: a PEGylated lipid; a helper phospholipid; a sterol; and a nucleic acid, wherein:(a) a ratio of the ionizable lipid to the helper phospholipid ranging from about 1 to about 200;(b) a ratio of the sterol to the PEGylated lipid ranging from about 10 to about 500;(c) a combined percentage of the ionizable lipid and the helper phospholipid ranging from about 20% to about 80%;(d) a ratio (N / P) of amine groups (N) of the ionizable lipid to phosphate groups (P) of the nucleic acid ranging from about 4 to about 15; and(e) wherein the lipid nanoparticle is capable of migrating to one or more systemic organs following intramuscular administration at a fraction ranging from about 10% to about 99% of an administered dose and mediates detectable expression of the nucleic acid in at least one systemic organ.

2. The composition of claim 1, wherein the ionizable lipid comprises ALC-0315.

3. The composition of claim 1, wherein the PEGylated lipid comprises DMG-PEG2000.

4. The composition of claim 1, wherein the helper phospholipid is selected from an anionic phospholipid, a zwitterionic phospholipid, and a cationic phospholipid.

5. The composition of claim 4, wherein the anionic phospholipid is selected from bis(monooleoylglycero)-phosphate (18BMP) and l-stearoyl-2-oleoyl-sn-glycero-3-phospho-(l’-rac-glycerol) (18PG).

6. The composition of claim 4, wherein the zwitterionic phospholipid is selected from 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).8243985.601_P18418-027. The composition of claim 6, wherein the zwitterionic phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

8. The composition of claim 4, wherein the cationic phospholipid is selected from 1.2-dioleoyl-3-trimethylammonium-propane (DOTAP) and dimethyl dioctadecyl ammonium (DDAB).

9. The composition of any one of claims 1 to 8, wherein the sterol is cholesterol.

10. The composition of claim 1, wherein:(a) the ionizable lipid has a molar percentage ranging from about 10% to about 60%; (b) the helper phospholipid has a molar percentage ranging from about 0% to about 40%; (c) the sterol has a molar percentage ranging from about 40% to about 90%;(d) the PEGylated lipid has a molar percentage ranging from about 0.1% to about 5%; and (e) an N: P ratio ranging from about 4 to about 15.

11. The composition of claim 10, wherein the lipid nanoparticle comprises:(a) a molar percentage of about 20% to about 30% ionizable lipid, about 20% to about 30% helper phospholipid, about 40% to about 55% sterol, about 1.5 to about 3.5% PEGylated lipid, and an N: P ratio of between about 8 to about 12;(b) a molar percentage of about 10% to about 20% ionizable lipid, about 1% to about 10% helper phospholipid, about 70% to about 90% sterol, about 0.1% to about 2% PEGylated lipid, and an N: P ratio of between about 8 to about 12;(c) a molar percentage of about 35% to about 55% ionizable lipid, about 5% to about 15% helper phospholipid, about 30% to about 50% sterol, about 0.1% to about 2.5% PEGylated lipid, and an N: P ratio of between about 4 to about 8;(d) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0 % PEGylated lipid, and an N: P ratio of between about 8 to about 10; or8343985.601_P18418-02(e) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1 to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0% PEGylated lipid, and an N: P ratio of between about 5 to about 9.

12. The composition of claim 11, wherein the lipid nanoparticle comprises:(a) a molar percentage of about 25% ionizable lipid, about 25% helper phospholipid, about 47.5% sterol, about 2.5% PEGylated lipid, and an N: P ratio of about 10;(b) a molar percentage of about 14% ionizable lipid, about 6% helper phospholipid, about 79.2% sterol, about 0.8% PEGylated lipid, and an N: P ratio of about 10;(c) a molar percentage of about 46.3% ionizable lipid, about 9.4% helper phospholipid, 42.7% sterol, about 1.6% PEGylated lipid, and an N: P ratio of about 6;(d) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% sterol, about 0.5% PEGylated lipid, and an N: P ratio of about 10; or(e) a molar percentage of about 45% ionizable lipid, about 5% helper phospholipid, about 49.5% sterol, about 0.5% PEGylated lipid, and an N: P ratio of about 7.

13. The composition of claim 10, wherein the lipid nanoparticle comprises:(a) a molar percentage of about 30% to about 40% ionizable lipid, about 10% to about 20% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 1.0% PEGylated lipid, and an N: P ratio of between about 5 and 9;(b) a molar percentage of about 35% to about 55% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 5 and about 9;(c) a molar percentage of about 40% to about 50% ionizable lipid, 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 2 to about 6;(d) a molar percentage of about 40% to about 50% ionizable lipid, about 0.1% to about 10% helper phospholipid, about 40% to about 60% sterol, about 0.1% to about 0.5% PEGylated lipid, and an N: P ratio of between about 8 to about 10;8443985.601_P18418-02(e) a molar percentage of about 30% to about 40% ionizable lipid, about 10% to about 20% helper phospholipid, about 40% to about 60% sterol, about 0.1 to about 1.0% PEGylated lipid, and an N: P ratio of between about 2 to about 6; or(f) a molar percentage of about 60% to about 80% ionizable lipid, about 5% to about 10% helper phospholipid, about 15% to about 25% sterol, about 0.01% to about 0.1% PEGylated lipid, and an N: P ratio of between about 8 to about 12.

14. The composition of claim 13, wherein the lipid nanoparticle comprises:(a) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% sterol, 0.5% PEGylated lipid, and an N: P ratio of about 7;(b) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 7;(c) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 4;(d) a molar percentage of about 45% ionizable lipid, 5% helper phospholipid, 49.9% sterol, 0.1% PEGylated lipid, and an N: P ratio of about 10;(e) a molar percentage of about 35% ionizable lipid, 15% helper phospholipid, 49.5% sterol, 0.5% PEGylated lipid, and an N: P ratio of about 4; or(f) a molar percentage of about 72% ionizable lipid. 8% helper phospholipid, 19.96% sterol, 0.04% PEGylated lipid, and an N: P ratio of about 10.

15. The composition of any one of claims 1 to 14, wherein the nucleic acid is selected from an antisense oligonucleotide, cDNA, genomic DNA, guide RNA, plasmid DNA (pDNA), vector DNA, mRNA, miRNA, piRNA, shRNA, and siRNA.

16. The composition of claim 15, wherein the nucleic acid is mRNA.

17. The composition of claim 15, wherein the nucleic acid comprises a polynucleotide encoding a Cas nuclease.

18. The composition of claim 17, wherein the Cas nuclease comprises Cas9.8543985.601_P18418-0219. The composition of any one of claims 17 to 18, wherein the lipid nanoparticle further comprises a small guide RNA or a DNA encoding a small guide RNA.

20. A vaccine comprising a lipid nanoparticle of the composition of any one of claims 1 to 19.

21. A method for treating a disease, condition, or disorder, the method comprising administering a composition of any one of claims 1 to 19 or a vaccine of claim 20 to a subject in need of treatment thereof.

22. The method of claim 21, wherein administration of the composition or the vaccine elicits a specific T cell response and / or cellular immunity.

23. The method of claim 21 or claim 22, wherein administration of the composition or the vaccine elicits a tissue-specific immune response at the primary site of infection.

24. The method of any one of claims 21 to 23, wherein the disease, condition, or disorder comprises a disease, condition, or disorder of the lung.

25. The method of claim 24, wherein the disease, condition, or disorder of the lung is selected from pneumonia, respiratory syncytial virus (RSV), influenza, tuberculosis, and a coronavirus.

26. The method of claim 21, wherein the disease, condition, or disorder comprises a cancer.

27. The method of claim 26, wherein administering the composition or the vaccine prevents or reduces one or more cancer cells remaining from a post-tumor excision or other surgical cancer treatment from metastasizing to one or more other organs.8643985.601_P18418-0228. The method of claim 21, wherein the disease, condition, or disorder comprises an infectious disease.

29. The method of claim 28, wherein the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the liver.

30. The method of claim 29, wherein the disease, condition, or disorder of the liver is selected from malaria, hepatitis B, and hepatitis C.

31. The method of any one of claims 28 to 30, wherein administration of the composition or the vaccine achieves gene expression in selected peripheral tissues and a lower systemic toxicity.

32. The method of claim 28, wherein the infectious disease, condition, or disorder comprises a disease, condition, or disorder of the lymphatic system.

33. The method of claim 32, wherein the disease, condition, or disorder of the lymphatic system comprises human immunodeficiency virus (HIV) / acquired immunodeficiency syndrome (AIDS).

34. The method of any one of claims 28 to 33, wherein tissue-resident T cells clear pathogen-invading cells.

35. The method of any one of claims 21 to 34, wherein the method is a prophylactic method of treatment.

36. The method of any one of claims 21 to 35, wherein the administering of the composition or the vaccine is via intramuscular (i.m.) injection or subcutaneous (s.c.) injection.8743985.601_P18418-02