Ionizable lipid prodrug compounds and lipid nanoparticles (LNPS) comprising same

Abstract

The present disclosure relates, in one aspect, to ionizable lipid prodrug compounds of formula (I). In another aspect, the disclosure relates to lipid nanoparticles (LNPs) comprising at least one ionizable lipid of formula (I). In another aspect, the disclosure provides methods of the LNPs of the disclosure for splenic delivery of therapeutic cargo.

Description

[0001] Attorney Docket No. 046483-7487W01(04056)

[0002] TITLE OF THE INVENTION

[0003] Ionizable Lipid Prodrug Compounds and Lipid Nanoparticles (LNPs) Comprising Same

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U. S. C. § 119(e) to U. S. Provisional Patent Application No. 63 / 710,328. filed October 22, 2024, which is incorporated herein by reference in its entirety.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0006] This invention was made with government support under TR002776 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] Messenger RNA (mRNA)-based cancer immunotherapy has emerged as a potential cancer treatment with remarkable progress over the last few years. Such therapies employ cytotoxic T cells which are critical for tumor surveillance. However, cancer is associated with T cell exhaustion, limiting tumor treatments to often fall short of their intended effectiveness. While the combination of mRNA with immune checkpoint blockade (ICB) therapies can inhibit T cell exhaustion and induce tumor regression, treatment resistance and low ICB response rates make them effective only in a subset of patients. This limitation has driven widespread interest in the development of immunotherapy platforms that target different immunosuppressive pathways.

[0009] There is thus a need in the art for compositions and methods of simultaneously targeting more than one immunosuppressive pathway. The present disclosure addresses this unmet need.

[0010] BRIEF SUMMARY OF THE INVENTION

[0011] In one aspect, the disclosure provides a compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:

[0012]

[0013] wherein: Attorney Docket No. 046483-7487W01(04056)

[0014]

[0015] e drug;

[0016] each occurrence of L1is independently selected from the group consisting of -O-, -N(RA)-, and -C(=O)-, and -(optionally substituted C1-C3 alkylenyl)-;

[0017] each occurrence of L2is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted Cs-Cs cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, -(optionally substituted C2-C8 heteroarylenyl)-X-, and -X-;

[0018] each occurrence of X, if present, is independently selected from the group consisting of -N(R2c)-, -N(R3)-, -N(RA)-, -C(=O)-, and -O-;

[0019] R1is H;

[0020] R2a, R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of optionally substituted C1-C24 alkyl and optionally substituted C1-C24 heteroalkyl;

[0021] R2d

[0022] - - {L3^-N^

[0023] each occurrence of R3is independently R2e;

[0024] each occurrence of L3is independently selected from the group consisting -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-Y-, -(optionally substituted C2-C12 alkenylenyl)-Y-, -(optionally substituted C1-C12 alkynylenyl)-Y-, -(optionally substituted C1-C12 heteroalkylenyl)-Y-, -(optionally substituted C3-C8 cycloalkylenyl)-Y-, -(optionally substituted C2-C8 heterocyloalkylenyl)-Y-, -(optionally substituted C6-C10 arylenyl)-Y-, - Attorney Docket No. 046483-7487W01(04056)

[0025] (optionally substituted C'2-Cs heteroarylenyl)-Y-, and -Y-;

[0026] each occurrence of Y, if present, is independently selected from the group consisting of -N(RA)-, -O-, and -C(=O)-;

[0027] m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0028] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0029] o is 1, 2, 3, 4. 5, 6, 7, 8, 9, or 10;

[0030] each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl; and

[0031] * indicates a bond between a C(=O) carbon and a nitrogen atom of

[0032]

[0033] .

[0034] In another aspect, the disclosure provides a lipid nanoparticle (LNP) composition comprising:

[0035] (a) at least one ionizable lipid;

[0036] (b) at least one neutral lipid;

[0037] (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof, wherein the LNP further comprises:

[0038] (i) at least one nucleic acid encoding an immunomodulatory' protein; and

[0039] (ii) at least one small molecule anticancer agent, wherein the small molecule anticancer agent is attached to at least one component of the LNP by a cleavable linker.

[0040] In certain embodiments, the immunomodulatory protein is interleukin 12 (IL-12) and the small molecule anticancer agent is indoximod.

[0041] In another aspect, the disclosure provides a lipid nanoparticle (LNP) composition comprising

[0042] (a) at least one ionizable lipid comprising the compound of formula (I);

[0043] (b) at least one neutral lipid;

[0044] (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof, wherein the LNP further comprises at least one nucleic acid encoding an immunomodulatory protein.

[0045] In another aspect, the disclosure provides a pharmaceutical composition comprising the LNP of the disclosure and at least one pharmaceutically acceptable carrier. Attorney Docket No. 046483-7487W01(04056)

[0046] In another aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises administering to the subject at least one LNP of the disclosure or a pharmaceutical composition thereof.

[0047] BRIEF DESCRIPTION OF THE FIGURES

[0048] The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

[0049] FIGs. 1 A-1B: Engineering IDO inhibitor-based prodrug ionizable lipids to facilitate IL-12 mRNA delivery via prodrug LNPs for synergistic cancer immunotherapy. FIG. 1A: Overview of the synthesis route, including the seven prodrug amine heads and three epoxide tails used to synthesize prodrug ionizable lipids (PILs). The disulfate linkage in the PIL can be triggered by intracellular GSH to release IDO inhibitor into cytoplasm. FIG. IB: A scheme showing the intra-tumoral injection of prodrug LNPs (PLNPs) which are composed of PILs, phospholipid, DMG-PEG, cholesterol, and IL- 12 mRNA. PLNPs induce T cell activation and suppress T cell exhaustion through IL- 12 expression and IDO pathway inhibitor release, respectively, thereby eliminating tumors through synergistic methods.

[0050] FIGs. 2A-2G: In vitro and in vivo screening of PIL library' and lead PLNPs for mRNA delivery. FIG. 2A: Scheme demonstrating screening method for PILs. PLNPs were formulated with a PIL. cholesterol, DOPE, and DMG-PEG at a molar ratio of 50 / 38.5 / 10 / 1.5. MC38 cells were used for in vitro screening of PLNPs encapsulating FLuc mRNA. Lead PLNPs were selected for in vivo evaluation via MC38 intra-tumoral administration. FIG. 2B: Heatmap displaying luminescence intensities of MC38 cells after treatment with PLNPs formulated from 21 distinct PILs. FIG. 2C: In vivo visualization of luminescence signals in mice after intra-tumoral administration of the top nine PLNPs, MC3 LNPs, or G0-6C-AA-C12 LNPs 6 h post-injection. FIG. 2D: In vivo FLuc expression at tumor tissue (n = 3 biologically independent samples). Mice were intratumorally injected with FLuc mRNA-loaded PLNPs, MC3 LNPs, or G0-6C-AA-C12 LNPs at an mRNA dosage of 2.5 pg per mouse for 6 h and the total flux was quantified. Data are presented as mean ± SD. FIG. 2E: Confocal images characterizing cellular uptake of DiO labeled G0-SS-AA-C12 PLNPs and MC3 LNPs in MC38 cells after 2 h of incubation. The cell nucleus and actin w ere stained by Hoechst 33342 and phalloidin, respectively. Scale bar: 20 pm. FIG. 2F: Representative confocal images demonstrating endosomal escape of G0-SS-AA-C12 PLNPs and MC3 LNPs. MC38 cells were treated with DiO-labelled LNPs for 2 h, then stained with Attorney Docket No. 046483-7487W01(04056)

[0051] LysoTracker Deep Red and Hoechst 33342 prior to imaging. Scale bar: 20 pm. FIG. 2G: Ex vivo FLuc expression in tumor tissue (n=3). Mice were intratumorally injected with G0-SS-AA-C10 / C12 / C14 FLuc pLNPs and MC3 FLuc LNP at a mRNA dosage of 2.5 pg per mouse for 6 h and the total flux was quantified. Data are presented as mean ± SD. Statistical differences were calculated using one-way ANOVA with Tukey’s multiple comparisons test.

[0052] FIGs. 3A-3P: Efficacy of G0-SS-AA-C12 for strong IL-12 mRNA transfection, and suppression of T cell exhaustion in vitro. FIG. 3A: Scheme depicting intracellular and extracellular IL-12 detection via ELISA. MC38 cells were treated with IL-12 mRNA encapsulated MC3 LNPs, G0-6C-AA-C12 LNPs, and G0-SS-AA-C12 PLNPs for 16 h at a dosage of 0.5 pg / mL per well. The supernatants and MC38 cells were collected for analysis via ELISA. FIG. 3B: Secreted IL-12 in cell growth medium 16 h after treatment (n = 4 biologically independent samples). FIG. 3C: IL-12 expression in lysed MC38 cells 16 h after incubation (n = 4 biologically independent samples). FIG. 3D: Representative flow cytometric histograms using PE-anti-IL12 antibody to characterize intracellular IL- 12 expression from MC38 cells treated with IL12 mRNA encapsulated within MC3 LNPs, G0-6C-AA-C12 LNPs, or G0-SS-AA-C12 PLNPs for 16 h at adosage of0.5 pg / mL per well. FIG. 3E: Quantification of flow cytometry-based IL- 12 expression in MC38 cells (n = 3 biologically independent samples). FIG. 3F: Immunofluorescence images of MC38 cells treated with different LNPs. MC3, G0-6C-AA-C12 LNPs and G0-SS-AA-C12 PLNPs delivering mRNA encoding IL-12 were co-cultured with MC38 cells for 16 h. Then, cells were subjected to an immunofluorescence assay. Scale bar: 20 pm. FIG. 3G: Mean fluorescence intensity7of anti-mouse IL-12-PE in MC38 cells after treated by MC3 IL-12, G0-6C-AA-C12 IL-12 LNPs, and G0-SSAA- C12 IL-12 PLNPs. FIG. 3H: Scheme depicting release of indoximod (1-methyl-D-try ptophan) from G0-SS-AA-C12 PLNPs triggered by GSH. FIG. 3I: Corresponding indoximod (1-methyLD-tryptophan) release curve after GSH triggering at different time points. FIG. 3J: Scheme depicting the workflow to characterize the effect of PLNP -mediated IL-12 secretion on T cell populations. MC38 cells were treated with G0-SS-AA-C12 IL-12 PLNPs or G0-SS-AA-C12 FLuc PLNPs for 24 h at a dose of 2 pg per well. Supernatants were collected and mixed with T cell culture-medium at a volume ratio of 1:1 and used to culture primary mouse CD8+ T cells for 24 h (PBS served as negative control). Then, T cells were harvested and subjected for flow cytometry analysis. FIG. 3K: Representative flow cytometric histograms after PD-1 antibody staining. FIG. 3L:

[0053] Quantification of PD-1 expression in CD8+ T cells after treatment with different groups (n = 3 biologically independent samples). Data are presented as mean ± SD. FIG. 3M: Workflow Attorney Docket No. 046483-7487W01(04056)

[0054] to characterize the effect of pLNP-mediated indoximod release and IL-12 secretion on T cell exhaustion and activation. FIGs. 3N-3P: Quantification of CD69 (FIG. 3N), CD25 (FIG. 30), and PD-1 expression (FIG. 3P) on OT-I CD3+T cells after treatment with different groups (n=4).

[0055] FIGs. 4A-4M: G0-SS-AA-C12 IL-12 PLNPs achieve therapeutic synergistic effects in vivo in an MC38 tumor model and induce a splenic T cell response. FIG. 4A: Schematic of evaluating PLNP-mediated anti-tumor effect. Mice were subcutaneously injected MC38 cells on Day 0. PBS, G0-SS-AA-C12 PLNPs encapsulating IL-12 mRNA, G0-6C-AA-C12 LNPs encapsulating IL-12 mRNA, or G0-SS-AA-C12 PLNPs encapsulating FLuc mRNA were intratumorally administrated on Day 6, Day 8. and Day 10. FIGs. 4B-4D: Tumor growth and body weight changes were monitored in different groups (n = 8 biologically independent mice per group). FIG. 4E: Survival curves for mice in each treatment group (n = 8 biologically independent mice per group). To assess if PLNPs treatment activated splenic T cells, the experiment described in FIG. 4A were repeated, but on Day 12, mice were euthanized and spleens were harvested and analyzed via flow cytometry. Representative flow dot plots (FIG. 4F) and corresponding statistical analysis (FIGs. 4G-4H) of CD8+ T cells and CD4+ T cells in spleen after treatment are shown. FIGs. 4I-4J: Quantification of effector memory CD8+ T cells and effector memory7CD4+ T cells in spleen. FIGs. 4K-4L:

[0056] Quantification of central memory CD8+ T cells and central memory CD4+ T cells in spleen. FIG. 4M: Quantification of Treg cells in spleen. Data are presented as mean ± SD.

[0057] FIGs. 5 A-5P: Characterization of tumor microenvironment in vivo after treatment with G0-SSAA IL-12 LNPs encapsulating IL-12 mRNA. FIG. 5A: Schematic showing establishment of MC38 tumor model for flow cytometry7analysis and bulk RNA sequencing after treatment. Mice bearing MC38 tumors received intra-tumoral injection of PBS, GO-SS-AA-C12 FLuc PLNPs, G0-6C-AAC12 IL-12 LNPs, or G0-SS-AA-C12 IL-12 PLNPs. On Day 12, tumors in each treatment group w ere collected, and a portion of tumor tissues w as digested into single-cell suspension for flow cytometry -based characterization of T cells, while the remaining cells were digested for RNA sequencing of tumor tissue. FIGs. 5B-5C:

[0058] Quantification of CD8+ T cells and CD4+ T cells in tumor tissue after different treatments. FIGs. 5D-5E: Analysis of PD-1 expression on the surface of CD8+ T cells and CD4+ T cells. FIG. 5F: IFN-y gene expression after treatment with G0-6C-AA-C12 IL- 12 LNPs and G0-SS-AA-C12 IL-12 PLNPs relative to PBS treatment. FIG. 5G: Summary of selected significant upregulation expressed genes in response to treatment with G0-SS-AA-C12 FLuc PLNPs, GO- 6C-AA-C12 IL-12 LNPs, and G0-SS-AA-C12 IL-12 PLNPs (n = 3 biologically Attorney Docket No. 046483-7487W01(04056)

[0059] independent samples). FIGs. 5H-5M: Heat maps of differentially expressed genes in response to treatment with PBS or G0-SS-AA-C12 IL-12 PLNPs. Data are presented as mean ± SD. FIGs. 5N-5P: Representative flow dot plots (FIG. 5P) and corresponding statistical analysis (FIGs. 5N-5O) of CD8+T cells (FIG. 5N and FIG. 5P) and CD4+T cells (FIG. 50 and FIG.

[0060] 5P) in the spleen after treatment are shown (n=5).

[0061] FIGs. 6A-6L: Intra-tumoral injection of G0-SS-AA-C12 IL-12 PLNPs elicits effective long-term antitumor response and drives regression of distal tumors. FIG. 6A: Scheme depicting in vivo MC38 tumor rechallenge. Mice bearing -100 mm3 MC38 tumors were injected with G0-SS-AAC12 IL-12 PLNPs at Day 6, Day 8, and Day 10, resulting in tumor elimination by Day 30. Then, MC38 cells were subcutaneously reinjected into the same position to monitor tumor growth and mouse survival. Mice naive to PLNPs treatment served as controls. FIG. 6B: Mean tumor volume from two independent experiments (n = 5 biologically independent samples). FIG. 6C: Spider plots of individual tumor growth curves in treatment group and control group. FIG. 6D: Survival curve of mice for treatment group in comparison to the control group (n = 5 biologically independent samples). FIG. 6E: Body w eight changes of mice in treatment group and control group. FIG. 6F: Scheme depicting the bilateral tumor model. Mice were treated with PBS, G0-SS-AA-C12 FLuc PLNPs, G0-6C-AA- C12 IL-12 LNPs, or G0-SS-AA-C12 IL-12 PLNPs in only the right tumor. Tumor growth and mouse survival in each group were monitored. FIG. 6G: Representative images of tumors on Day 30 after initiation of treatment. FIG. 6H: Spider plots of individual tumor growth curves in different treatment groups. FIG. 6I: Survival curves from the four different treatment groups. FIG. 6J: Body w eight changes of mice in treatment group and control group. FIGs. 6K-6L: Quantification of CD8+ T cells and CD4+ T cells in contralateral tumor tissue after treatment. Data are presented as mean ± SD.

[0062] FIG. 7: Synthetic routes for prodrug ionizable lipids.

[0063] FIG. 8: Chemical structures of prodrug amine heads.

[0064] FIG. 9: Chemical structures of 21 prodrug ionizable lipids.

[0065] FIG. 10: Luminescence level of HepG2 cells following treating with FLuc mRNA-loaded LNPs at an mRNA dose of 62.5 ng / well for 8 h.

[0066] FIG. 11: In vivo bioluminescence imaging at 6 h post-treatment of FLuc-loaded LNPs. Mice were i.v. injected with FLuc-loaded LNPs (2.5 pg mRNA per mouse). Total flux at liver were quantified.

[0067] FIG. 12: MC38 cell viability after treating with LNPs encapsulating FLuc mRNA. Cells were treated with FLuc-loaded LNPs for 8 h. Results were normalized to untreated cells Attorney Docket No. 046483-7487W01(04056)

[0068] (0 ng mRNA / well).

[0069] FIG. 13: Synthetic routes for G0-6C-AA-C12.

[0070] FIG. 14: Luminescence level of MC38 cells following treating with FLuc mRNA-loaded LNPs at an mRNA dose of 62.5 ng / well for 8 h.

[0071] FIG. 15 A: Cryo-TEM image of G0-SS-AA-C12 PLNPs encapsulating FLuc mRNA. Scale bar: 50 nm. FIG. 15B: DLS recorded for G0-SS-AA-C12 PLNPs. FIG. 15C: A table presenting physiochemical parameters of the G0-SS-AA-C12 PLNPs, including size, poly dispersity index (PDI), zeta potential, encapsulated efficiency (EE%), and pKa.

[0072] FIG. 16A: Cryo-TEM image of G0-6C-AA-C12 LNPs encapsulating FLuc mRNA. Scale bar: 50 nm. FIG. 16B: DLS recorded for G0-6C-AA-C12 LNPs. FIG. 16C: Atable presenting physiochemical parameters of the G0-6C-AA-C12 LNPs, including size, poly dispersity index (PDI), zeta potential, encapsulated efficiency (EE%), and pKa.

[0073] FIGs. 17A-17B: TNS assay was used to determine the apparent pKa of G0-SS-AA-C12 PLNPs (FIG. 17A) and G0-6C-AA-C12 LNPs (FIG. 17B). pKa is calculated as the pH corresponding to half of the maximum TNS fluorescence value.

[0074] FIG. 18: Confocal images characterizing cellular uptake of DiO labeled G0-6C-AA-C12 LNPs in MC38 cells after 2 h of incubation. PBS was used as negative control. The cell nucleus and actin were stained by Hoechst 33342 and phalloidin, respectively. Scale bar: 20 pm.

[0075] FIG. 19: HPLC traces recoded for 1-methyl-D-try ptophan (indoximod) at 1, 5, 10, 25, 50 ng / mL, respectively. HPLC conditions: MeCN / H2O with a gradient from 1 / 1 -9 / 1; detection wavelength: 286 nm.

[0076] FIG. 20: The standard calibration curve o IN -methyl - / / -try ptophan (indoximod). FIG. 21: HPLC traces recoded for monitoring I -methyl - / / -try ptophan release after GSH triggering at different time points. HPLC conditions: MeCN / H2O with a gradient from 1 / 1 -9 / 1; detection wavelength: 286 nm.

[0077] FIG. 22: The structures of G0-SS-AA-C12 FLuc PLNP, G0-6C-AA-C12 IL-12 LNP, and G0-SS-AA-C12 IL-12 PLNP, respectively.

[0078] FIG. 23: H& E-stained sections of major organs in MC38 tumor-bearing mice after treatment with different LNPs. Scale bar: 275 pm.

[0079] FIG. 24: Gating strategy for identification of spleen CD4+ and CD8+ T cells and the corresponding effector memory (CD44+CD62L-) and central memory' (CD44+CD62L+) CD4+ and CD8+ T cell populations.

[0080] FIGs. 25A-25B: The MC38 tumor bearing mice were treated with PBS, G0-SS-AA- Attorney Docket No. 046483-7487W01(04056)

[0081] C12 FLuc PLNPs, G0-6C-AA-C12 IL-12 LNPs, and G0-SS-AA-C12 IL-12 PLNPs via intratumor administration, respectively. Spleens were harvested for flow analysis.

[0082] Representative flow dot plots of spleen center memory (CD44+CD62L+) and effector (CD44+CD62L-) CD8+ and CD4+ T cells after different treatment.

[0083] FIG. 26: Gating strategy for identifying regulatory T cells in spleen.

[0084] FIG. 27: Representative flow dot plots of regulatory T cells in spleen after MC38 tumor bearing mice with treatment of PBS, G0-SS-AA-C12 FLuc PLNPs, G0-6C-AA-C12 IL-12 LNPs, and G0-SS-AA-C12 IL-12 PLNPs, respectively.

[0085] FIG. 28: Gating strategy for identification of infiltrated CD4+ and CD8+ T cells and the corresponding CD4+PD-1+ and CD8+PD-1+ T cell populations in the tumor tissue.

[0086] FIGs. 29A-29C: Representative flow dot plots of infiltrated CD4+ and CD8+ T cells (FIG. 29 A) and corresponding CD8+PD-1+ (FIG. 29B) and CD4+PD-1+ (FIG. 29C) T cell populations in the tumor tissue after MC38 tumor bearing mice treated with PBS, GO-SS-AA-FLuc PLNPs, G0-6C-AA-C12 IL- 12 LNPs, and G0-SS-AA-C12 IL-12 PLNPs, respectively.

[0087] FIG. 30A: Volcano plots of changes in gene expression (log2) between MC38 tumors treated with PBS and G0-SS-AA-C12 IL-12 PLNPs. FIG. 30B: Gene set enrichment analysis (GSEA) of MC38 tumors when treated with G0-SS-C12 IL-12 PLNPs.

[0088] FIG. 31 A: Volcano plots of changes in gene expression (log2) between MC38 tumors treated with PBS and G0-SS-AA-C12 FLuc PLNPs. FIGs. 31B-31D: Heat maps of differentially expressed genes in response to treatment with PBS or G0-SS-AA-C12 FLuc PLNPs.

[0089] FIG. 32A: Volcano plots of changes in gene expression (log2) between MC38 tumors treated with PBS and G0-6C-AA-C12 IL-12 LNPs. FIGs. 32B-32D: Heat maps of differentially expressed genes in response to treatment with PBS or G0-6C-AA-C12 IL- 12 LNPs.

[0090] FIG. 33: Representative flow dot plots of infiltrated CD4+ and CD8+ T cells in contralateral tumor tissue. Mice with bilateral tumors were treated with PBS, G0-SS-AA-C12 FLuc PLNPs, G0-6C-AA-C12 IL-12 LNPs, and G0-SS-AA-C12 IL-12 PLNPs, respectively, in only the right tumor. The left tumor was harvested for flow analysis to characterize the infiltrated CD4+ and CD8+ T cells.

[0091] FIGs. 34A-34D: Evaluation of the pLNP-mediated anti-tumor efficacy after intravenous administration. FIG. 34A: Mice were subcutaneously injected with MC38 cells on day 0. PBS. free mRNA, free IDO inhibitor, G0-SS-AA-C12 FLuc pLNP. G0-6C-AA-C12 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor, and G0-SS-AA-C12 IL-12 Attorney Docket No. 046483-7487W01(04056)

[0092] pLNP were intravenously administered on day 6, day 8, and day 10. Tumor growth and mice survival were monitored. FIGs. 34B-34D: Tumor growth and mice survival were monitored. FIG. 34B provides a graph. FIG. 34C provides graphs. FIG. 34D provides a graph. Data are presented as mean ± SD. Statistical differences in FIG. 34B were calculated using two-way ANOVA with Tukey’s multiple comparisons test. Survival analysis in FIG. 34D was performed using a log-rank (Mantel-Cox) test (n=5 biologically independent samples).

[0093] FIGs. 35A-35J: Evaluation of the systemic toxicity of pLNPs after intravenous (i.v.) injection or intratumoral (i.t.) injection. FIG. 35A: Mice were subcutaneously injected with MC38 cells on day 0. Free IL-12 mRNA, free IDO inhibitor, G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor, or GO-SS-AA-C12 IL-12 pLNP were administered either i.v. or i.t. on day 6, day 8. and day 10. PBS served as negative control. The serum of mice from each treatment group was collected on day 12. FIGs. 35B-35G: IL-6, TNF-a+, and IFN-y+ levels in serum after i.v. or i.t. injection of each treatment. FIG. 35B: IL-6 in serum before injection. FIG. 35C: TNF-a in serum before injection. FIG. 35D: IFN-y in serum before injection. FIG. 35E: IL-6 in serum after injection. FIG. 35F: TNF-a in serum after injection. FIG. 35G: IFN-y in serum after injection. FIGs. 35H-35J: Comparison of IL-6 (FIG. 35H), TNF-a+ (FIG. 35I), and IFN-y+ (FIG. 35 J) levels in serum after i.v. or i.t. injection of G0-SS-AA-C12 IL-12 pLNP. Data are presented as mean ± SD. Statistical differences were calculated using one-way ANOVA with Tukey’s multiple comparisons test (n=3 biologically independent samples).

[0094] FIG. 36: AST and ALT levels after intravenous injection of pLNP and control therapeutics. Tumor-bearing mice were intravenously injected with free IL- 12 mRNA, free IDO inhibitor, G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor, or G0-SS-AA-C12 IL- 12 pLNP. PBS was injected as a negative control. AST and ALT levels were quantified via ELISA. Data are presented as mean ± SD. Statistical differences were calculated using one-way ANOVA with Tukey’s multiple comparisons test (n=3 biologically independent samples).

[0095] FIG. 37: AST and ALT levels after intratumoral injection of pLNP and control therapeutics. Tumor-bearing mice were intratumorally injected with free IL-12 mRNA, free IDO inhibitor, G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL- 12 LNP, G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor, or G0-SS-AA-C12 IL-12 pLNP. PBS was injected as a negative control. AST and ALT levels were quantified via ELISA. Data are presented as mean ± SD. Statistical differences were calculated using one-way ANOVA with Tukey’s multiple comparisons test (n=3 biologically independent samples). Attorney Docket No. 046483-7487W01(04056)

[0096] DETAILED DESCRIPTION OF THE INVENTION

[0097] Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

[0098] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include notjust about 0.1% to about 5%, but also the individual values (e.g, 1%, 2%, 3%, and 4%) and the sub-ranges (e.g, 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

[0099] In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting: information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

[0100] In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process. Attorney Docket No. 046483-7487W01(04056)

[0101] Description

[0102] As indicated elsewhere herein, limitations associated with T cell exhaustion, inter alia, associated with current mRNA-based cancer immunology methods have driven interest in the development of immunotherapy platforms that target different immunosuppressive pathways. The highly expressed enzyme indoleamine 2,3-dioxygenase (IDO) in cancer cells which involves in tryptophan catabolism is a common and important therapeutic target. The over-expressed IDO induces the exhaustion and apoptosis of effector T cells and facilitates cancer immune evasion by depleting tryptophan levels and accumulating kynurenines (Kyn), yielding immunosuppression in the tumor microenvironment (TME). One specific IDO inhibitor, the small molecule drug indoximod ( I -methyl- / Mry ptophan). is currently being evaluated in clinical trials for the treatment of solid tumors. Indoximod inhibits the conversion of tryptophan to Kyn and promotes a more favorable TME to limit T cell exhaustion and enhance T cell antitumor activity. Therefore, targeting IDO and combining it with mRNA could be a potential approach to improve mRNA-based immunotherapies for a range of solid tumors.

[0103] Additionally, activating native T cells via cytokine-based immunotherapy has become a central point of many early clinical trials for diverse malignancies. One of these cytokines, interleukin- 12 (IL-12) holds great promise for stimulating the effector functions of T cells, given its major role in enhancing T cell proliferation and activating tumor-specific cytotoxic T cells. However, systemic administration of IL- 12 is poorly tolerated, rapidly increasing pro-inflammatory cytokines and potentially inducing a life-threatening cytokine storm. Thus, there is a clear need of a more tumor-specific and transient method for the delivery' of IL- 12.

[0104] The combination of local IL-12-mediated T cell activation w ith IDO inhibitor-mediated reversal of T cell exhaustion may be an exciting synergistic strategy’ for cancer immunotherapy. Leveraging the features of local and transient translation of mRNA, codelivery of these therapeutic molecules may’ be achieved via direct intra-tumoral administration of lipid nanoparticles (LNPs) encapsulating mRNA. LNPs are composed of ionizable lipids (ILs), phospholipids, cholesterol, and polyethylene glycol (PEG)-conjugated lipids. While LNPs are highly effective for mRNA delivery, the loading efficiency of free small molecule drugs in LNPs is low and has the risk of drug leakage in vivo. In contrast, prodrug-based nanosystems have higher drug loading capacity’, good biocompatibility’, and better controlled drug release in vivo. Thus, ILs conjugated to indoximod were engineered, generating prodrug ILs (pILs) that could be used to formulate prodrug LNPs (pLNPs) encapsulating IL- 12 mRNA for combination solid tumor immunotherapy. Attorney Docket No. 046483-7487W01(04056)

[0105] To this end. a library of prodrug ionizable amines were synthesized that possess indoxi mod. reactive amine groups, and a glutathione (GSH)-cleavable disulfide linkage (FIG.

[0106] 1 A). These prodrug amine heads were reacted with epoxide-terminated lipid tails to generate a library of pILs that were incorporated into chemically distinct pLNPs (FIG. IB). In vitro and in vivo screening of pLNPs encapsulating reporter firefly luciferase (FLuc) mRNA revealed a top-performing pIL, G0-SS-AA-C12. Substitution of FLuc mRNA with IL-12 mRNA in G0-SS-AA-C12 pLNP resulted in both strong IL- 12 transfection of cancer cells and indoximod release (FIG. 1 A), leading to T cell activation and reversal of T cell exhaustion (FIG. 1C). In aMC38 colon adenocarcinoma mouse model, G0-SS-AA-C12 pLNP encapsulating IL- 12 mRNA boasted excellent anti -tumor efficacy, owing to the synergistic effect of both therapeutic cargos. Moreover. G0-SS-AA-C12 pLNP encapsulating IL-12 mRNA demonstrated protection against tumor rechallenge and drove distal antitumor efficacy in a bilateral tumor model. This study highlights the therapeutic potential of a small molecule-conjugated mRNA-LNP for cancer immunotherapy and offers a potentially broadly applicable strategy to generate a range of small molecule-RNA platforms for cancer therapeutic applications.

[0107] Definitions

[0108] The term “about" ’ as used herein can allow for a degree of variability in a value or range, for example, within 10%. within 5%. or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

[0109] The term “alkenyl’’ as used herein refers to straight and branched chain and cyclic alkyl groups as defined herein, except that at least one double bond exists between two carbon atoms. Thus, alkenyl groups have from 2 to 40 carbon atoms, or 2 to about 20 carbon atoms, or 2 to 12 carbon atoms or, in some embodiments, from 2 to 8 carbon atoms.

[0110] Examples include, but are not limited to vinyl, -CH=C=CCH2, -CH=CH(CH3), -CH=C(CH3)2, -C(CH3)=CH2, -C(CH3)=CH(CH3), -C(CH2CH3)=CH2, cyclohexenyl, cyclopentenyl, cyclohexadienyl, butadienyl, pentadienyl, and hexadienyl among others.

[0111] The term “alkoxy” as used herein refers to an oxygen atom connected to an alkyl group, including a cycloalkyl group, as are defined herein. Examples of linear alkoxy groups include but are not limited to methoxy, ethoxy, propoxy, butoxy, penty loxy, hexyloxy, and the like. Examples of branched alkoxy include but are not limited to isopropoxy, sec-butoxy. tert-butoxy, isopentyloxy, isohexyloxy, and the like. Examples of cyclic alkoxy include but are not limited to cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy, and the Attorney Docket No. 046483-7487W01(04056)

[0112] like. An alkoxy group can include about 1 to about 12, about 1 to about 20, or about 1 to about 40 carbon atoms bonded to the oxygen atom, and can further include double or triple bonds, and can also include heteroatoms. For example, an allyloxy group or a methoxyethoxy group is also an alkoxy group within the meaning herein, as is a methylenedioxy group in a context where two adjacent atoms of a structure are substituted therewith.

[0113] The term “alkyl” as used herein refers to straight chain and branched alkyl groups and cycloalkyl groups having from 1 to 40 carbon atoms, 1 to about 20 carbon atoms, 1 to 12 carbons or, in some embodiments, from 1 to 8 carbon atoms. Examples of straight chain alkyl groups include those with from 1 to 8 carbon atoms such as methyl, ethyl, n-propyl, n-butyl, n-penty l, n-hexyl, n-heptyl, and n-octyl groups. Examples of branched alkyl groups include, but are not limited to, isopropyl, iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and 2,2-dimethylpropyl groups. As used herein, the term “alkyl” encompasses n-alkyl, isoalkyl, and anteisoalkyl groups as well as other branched chain forms of alkyl. Representative substituted alkyl groups can be substituted one or more times with any of the groups listed herein, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups.

[0114] The term “alkynyl” as used herein refers to straight and branched chain alkyl groups, except that at least one triple bond exists between two carbon atoms. Thus, alkynyl groups have from 2 to 40 carbon atoms, 2 to about 20 carbon atoms, or from 2 to 12 carbons or, in some embodiments, from 2 to 8 carbon atoms. Examples include, but are not limited to -C≡CH. -C≡CCH₃. -OC(CH2CH3), -CH2C=CH. -CH2CAC(CH3). and -CH2OC(CH2CH3) among others.

[0115] The term “alkylene” or “alkylenyl” as used herein refers to a bivalent saturated aliphatic radical (e.g, -CH2-, -CH2CH2-, and -CH2CH2CEI2-, inter alia). In certain embodiments, the term may be regarded as a moiety derived from an alkene by opening of the double bond or from an alkane by removal of two hydrogen atoms from the same (e.g., -CH2-) different (e.g, -CH2CH2-) carbon atoms. Similarly, the terms "heteroalkylenyl".

[0116] “cycloalkylenyl”, “heterocycloalkylenyl”, and the like, as used herein, refer to a divalent radical of the moiety corresponding to the base group (e.g, heteroalkyl, cycloalkyl, and / or heterocycloalkyl). A divalent radical possesses two open valencies at any position(s) of the group, wherein each radical may be on a carbon atom or heteroatom. Thus, the divalent radical may form a single bond to two distinct atoms or groups, or may form a double bond with one atom.

[0117] The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an Attorney Docket No. 046483-7487W01(04056)

[0118] adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an '“antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

[0119] The term “amine” as used herein refers to primary, secondary, and tertiary amines having, e.g., the formula N(group)?, wherein each group can independently be H or non-H, such as alkyl, aryl, and the like. Amines include but are not limited to R-NH2, for example, alky lamines, arylamines, alkylarylamines; R2NH wherein each R is independently selected, such as dialkylamines, diarylamines, aralkylamines, heterocyclylamines and the like; and R3N wherein each R is independently selected, such as trialkylamines, dialkylarylamines, alkyldiarylamines, triarylamines. and the like. The term “amine” also includes ammonium ions as used herein.

[0120] The term “amino group” as used herein refers to a substituent of the form -NH2, -NHR, -NR2, -NR, wherein each R is independently selected, and protonated forms of each, except for -NRV. which cannot be protonated. Accordingly, any compound substituted with an amino group can be viewed as an amine. An “amino group” within the meaning herein can be a primary, secondary, tertiary, or quaternary amino group. An “alkylamino” group includes a monoalkylamino, dialkylamino, and trialkylamino group.

[0121] The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N-succinylphosphatidylethanolamines. N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic Attorney Docket No. 046483-7487W01(04056)

[0122] modifying groups joined to neutral lipids.

[0123] The term “aryl” as used herein refers to cyclic aromatic hydrocarbon groups that do not contain heteroatoms in the ring. Thus aryl groups include, but are not limited to, phenyl, azulenyl, heptalenyl, biphenyl, indacenyl, fluorenyl, phenanthrenyl, triphenylenyl, pyrenyl, naphthacenyl, chrysenyl, biphenylenyl, anthracenyl, and naphthyl groups. In some embodiments, aryl groups contain about 6 to about 14 carbons in the ring portions of the groups. Aryl groups can be unsubstituted or substituted, as defined herein. Representative substituted aryl groups can be mono-substituted or substituted more than once, such as, but not limited to, a phenyl group substituted at any one or more of 2-, 3-, 4-, 5-, or 6-positions of the phenyl ring, or a naphthyl group substituted at any one or more of 2- to 8-positions thereof.

[0124] The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). It has been found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U. S. Patent Publication Nos.

[0125] 20060083780 and 20060240554; U. S. Patent Nos. 5,208,036; 5,264,618; 5,279,833;

[0126] 5,283,185; 5.753,613; and 5.785,992; and PCT Publication No. WO 96 / 10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cat-ionic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, Cis alky l chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA. DLinDMA, DLenDMA. and DODMA.

[0127] The term “cycloalkyl” as used herein refers to cyclic alkyl groups such as, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl groups. In some embodiments, the cycloalkyl group can have 3 to about 8-12 ring members, whereas in other embodiments the number of ring carbon atoms range from 3 to 4, 5, 6, or 7. Cycloalkyl groups further include polycyclic cycloalkyl groups such as, but not limited to, norbomyl, adamantyl, bomyl, camphenyl, isocamphenyl, and carenyl groups, and fused rings such as, but not limited to, decalinyl, and the like. Cycloalkyl groups also include rings that are substituted with straight or branched chain alkyl groups as defined herein. Representative substituted cycloalkyl groups can be mono-substituted or substituted more than once, such as, but not limited to, 2,2-, 2,3-, 2,4- 2,5- or 2,6-disubstituted cyclohexyl groups or mono-, di- or Attorney Docket No. 046483-7487W01(04056)

[0128] tri-substituted norbomyl or cycloheptyl groups, which can be substituted with, for example, amino, hydroxy, cyano, carboxy, nitro, thio, alkoxy, and halogen groups. The term “cycloalkenyl” alone or in combination denotes a cyclic alkenyl group.

[0129] A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.

[0130] In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

[0131] A disease or disorder is "alleviated” if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by a patient, or both, is reduced.

[0132] As used herein, the terms “effective amount,” “pharmaceutically effective amount” and “therapeutically effective amount” refer to a nontoxic but sufficient amount of an agent to provide the desired biological result. That result may be reduction and / or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An appropriate therapeutic amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation.

[0133] In particular, in the case of a mRNA, and “effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid as relating to a mRNA is an amount sufficient to produce the desired effect, e.g., mRNA-directed expression of an amount of a protein that causes a desirable biological effect in the organism within which the protein is expressed. For example, in some embodiments, the expressed protein is an active form of a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the protein that is normally expressed in the cell type of a healthy individual. For example, in some embodiments, the expressed protein is a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the mRNA is an amount that produces a similar level of expression as observed in a healthy individual in an individual with aberrant expression of the protein (z.e., protein deficient individual). Suitable assays for measuring the expression of an mRNA or protein include, but are not limited to dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well Attorney Docket No. 046483-7487W01(04056)

[0134] as phenotypic assays known to those of skill in the art.

[0135] The term “encode” as used herein refers to the product specified (e.g., protein and RNA) by a given sequence of nucleotides in a nucleic acid (z.e., DNA and / or RNA), upon transcription or translation of the DNA or RNA, respectively. In certain embodiments, the term “encode” refers to the RNA sequence specified by transcription of a DNA sequence. In certain embodiments, the term “encode” refers to the amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In certain embodiments, the term “encode” refers to the amino acid sequence specified by transcription of DNA to mRNA and subsequent translation of the mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may comprise a direct transcription or translation product. In certain embodiments, the encoded product may comprise post-translational modifications understood or reasonably expected by one skilled in the art.

[0136] The term “fully encapsulated” indicates that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particle is degraded in a treatment that would normally degrade 100% of free active agent or therapeutic agent, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particle is degraded. In the context of nucleic acid therapeutic agents, full encapsulation may be determined by an OLIGREEN® assay. OLIGREEN® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). RiboGreen is an ultra-sensitive fluorescent nucleic acid stain for quantitating RNA in solution. “Fully encapsulated” also indicates that the lipid particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

[0137] The terms “halo,” “halogen,” or “halide” group, as used herein, by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom.

[0138] The term “haloalkyl” group, as used herein, includes mono-halo alkyl groups, polyhalo alkyl groups wherein all halo atoms can be the same or different, and per-halo alkyl groups, wherein all hydrogen atoms are replaced by halogen atoms, such as fluoro. Examples of haloalkyl include trifluoromethyl. 1,1 -dichloroethyl, 1.2-di chloroethyl, l,3-dibromo-3,3-difluoropropyl, perfluorobutyl, and the like. Attorney Docket No. 046483-7487W01(04056)

[0139] The term “helper lipid'’ as used herein refers to a lipid capable of increasing the effectiveness of delivery of lipid-based particles such as cationic lipid-based particles to a target, preferably into a cell. The helper lipid can be neutral, positively charged, or negatively charged. In certain embodiments, the helper lipid is neutral or negatively charged. Nonlimiting examples of helper lipids include l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholin (POPC) and l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).

[0140] The term “heteroalkyl” as used herein by itself or in combination with another term, means, unless otherw ise stated, a non-cyclic stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) (e.g., O, N, P, and S) may be placed at any interior position of the heteroalkyl group or at either terminal position at which the group is attached to the remainder of the molecule.

[0141] The term “heteroaryl” as used herein refers to aromatic ring compounds containing 5 or more ring members, of which, one or more is a heteroatom such as, but not limited to, N, O, and S; for instance, heteroaryl rings can have 5 to about 8-12 ring members. A heteroaryl group is a variety of a heterocyclyl group that possesses an aromatic electronic structure. A heteroaryl group designated as a C2-heteroaryl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth.

[0142] Likewise a C-i-heteroaryl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms sums up to equal the total number of ring atoms. Heteroaryl groups include, but are not limited to, groups such as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Heteroaryl groups can be unsubstituted, or can be substituted with groups as is discussed herein. Representative substituted heteroaryl groups can be substituted one or more times with groups such as those listed herein.

[0143] Additional examples of ary l and heteroaryl groups include but are not limited to Attorney Docket No. 046483-7487W01(04056)

[0144] phenyl, biphenyl, indenyl, naphthyl (1-naphthyl, 2-naphthyl), N-hydroxytetrazolyl, N-hydroxytriazolyl, N-hydroxyimidazolyl. anthracenyl (1-anthracenyl, 2-anthracenyl, 3-anthracenyl), thiophenyl (2 -thienyl, 3-thienyl), furyl (2-furyl, 3-furyl), indolyl, oxadiazolyl, isoxazolyl, quinazolinyl, fluorenyl, xanthenyl, isoindanyl, benzhydryl. acridinyl, thiazolyl, pyrrolyl (2-pyrrolyl), pyrazolyl (3-pyrazolyl), imidazolyl (1-imidazolyl, 2 -imidazolyl, 4-imidazolyl, 5-imidazolyl), triazolyl (1,2,3-triazol-l-yl, l,2,3-triazol-2-yl l,2,3-triazol-4-yl, l,2,4-triazol-3-yl), oxazolyl (2-oxazolyl, 4-oxazolyl, 5-oxazolyl), thiazolyl (2 -thiazolyl, 4-thiazolyl, 5-thiazolyl), pyridyl (2-pyridyl, 3-pyridyl, 4-pyridyl), pyrimidinyl (2-pyrimidinyl, 4-pyrimidinyl, 5 -pyrimidinyl, 6-pyrimidinyl), pyrazinyl, pyridazinyl (3- pyridazinyl, 4-pyridazinyl, 5 -pyridazinyl), quinolyl (2-quinolyl, 3-quinolyl, 4-quinolyl, 5-quinolyl, 6-quinolyl. 7-quinolyl. 8-quinolyl), isoquinolyl (1 -isoquinolyl, 3-isoquinolyl. 4-isoquinolyl, 5-isoquinolyl, 6-isoquinolyl, 7-isoquinolyl 8-isoquinolyl), benzo[b]furanyl (2-benzo[b]furanyl, 3-benzo[b]furanyl, 4-benzo[b] furanyl, 5-benzo[b]furanyl, 6-benzo[b] furanyl, 7-benzo[b] furanyl), 2,3-dihydro-benzo[b]furanyl (2-(2,3-dihydro-benzo[b]furanyl), 3-(2,3-dihydro-benzo[b] furanyl), 4-(2,3-dihydro-benzo[b]furanyl), 5-(2,3-dihydro-benzo[b]furanyl), 6-(2,3-dihydro-benzo[b]furanyl), 7-(2,3-dihydro-benzo[b]furanyl), benzo|b]thiophenyl (2-benzo[b]thiophenyl, 3-benzo[b]thiophenyl, 4-benzo[b]thiophenyl, 5-benzo[b]thiophenyl, 6-benzo[b]thiophenyl, 7-benzo[b]thiophenyl), 2,3-dihydro-benzo[b]thiophenyl, (2-(2,3-dihydro-benzo[b] thiophenyl), 3-(2,3-dihydro-benzo[b]thiophenyl), 4-(2,3-dihydro-benzo[b]thiophenyl). 5-(2.3-dihydro-benzo[b]thiophenyl), 6-(2.3-dihydro-benzo[b]thiophenyl), 7-(2,3-dihydro-benzo[b]thiophenyl), indolyl (1 -indolyl, 2-indolyl, 3-indolyl, 4-indolyl, 5-indolyl, 6-indolyl, 7-indolyl), indazole (1-indazolyl, 3-indazolyl, 4-indazolyl, 5-indazolyl, 6-indazolyl, 7-indazolyl), benzimidazolyl (1 -benzimidazolyl, 2-benzimidazolyl, 4-benzimidazolyl, 5 -benzimidazolyl. 6-benzimidazolyl, 7-benzimidazolyl, 8-benzimidazolyl), benzoxazolyl (1-benzoxazolyl, 2-benzoxazolyl), benzothiazolyl (1-benzothiazolyl, 2-benzothiazolyl, 4-benzothiazolyl, 5-benzothiazolyl, 6-benzothiazolyl, 7-benzothiazolyl), carbazolyl (1-carbazolyl, 2-carbazolyl, 3-carbazolyl, 4-carbazolyl), 5H-dibenz[b.f] azepine (5H-dibenz[b,f] azepin- 1-yl, 5H-dibenz[b,f]azepine-2-yl,

[0145] 5H-dibenz[b,f]azepine-3-yl, 5H-dibenz[b,f]azepine-4-yl, 5H-dibenz[b,f]azepine-5-yl), 10,11 -dihy dro-5H-dibenz[b,f] azepine (10, 11 -dihydro-5H-dibenz[b,f] azepine-1 -yl,

[0146] 10,1 l-dihydro-5H-dibenz[b,f]azepine-2-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-3-yl, 10,1 l-dihydro-5H-dibenz[b,f]azepine-4-yl, 10,ll-dihydro-5H-dibenz[b,f|azepine-5-yl), and the like.

[0147] The term '‘heterocycloalkyl” as used herein refers to an aliphatic, partially unsaturated Attorney Docket No. 046483-7487W01(04056)

[0148] or fully saturated, 3- to 14-membered ring system, including single rings of 3 to 8 atoms and bi- and tricyclic ring systems where at least one of the carbon atoms of the ring is replaced with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. A heterocycloalkyl can include one to four heteroatoms independently selected from oxygen, nitrogen, and sulfur, wherein a nitrogen and sulfur heteroatom optionally can be oxidized and a nitrogen heteroatom optionally can be substituted. Representative heterocycloalkyl groups include, but are not limited, to the following exemplary groups: pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, and tetrahydrofuryl.

[0149] The term “heterocyclyl” as used herein refers to aromatic and non-aromatic ring compounds containing three or more ring members, of which one or more is a heteroatom such as, but not limited to, N, O, and S. Thus, a heterocyclyl can be a cycloheteroalkyl, or a heteroaryl, or if polycyclic, any combination thereof. In some embodiments, heterocyclyl groups include 3 to about 20 ring members, whereas other such groups have 3 to about 15 ring members. A heterocyclyl group designated as a C2-heterocyclyl can be a 5-ring with two carbon atoms and three heteroatoms, a 6-ring with two carbon atoms and four heteroatoms and so forth. Likewise a C4-heterocyclyl can be a 5-ring with one heteroatom, a 6-ring with two heteroatoms, and so forth. The number of carbon atoms plus the number of heteroatoms equals the total number of ring atoms. A heterocyclyl ring can also include one or more double bonds. A heteroaryl ring is an embodiment of a heterocyclyl group. The phrase “heterocyclyl group" includes fused ring species including those that include fused aromatic and non-aromatic groups. For example, a dioxolanyl ring and a benzdioxolanyl ring system (methylenedioxyphenyl ring system) are both heterocyclyl groups within the meaning herein. The phrase also includes polycyclic ring systems containing a heteroatom such as, but not limited to, quinuclidyl. Heterocyclyl groups can be unsubstituted, or can be substituted as discussed herein. Heterocyclyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl, benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl, indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl, isoquinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinyl groups. Representative substituted heterocyclyl groups can be mono-substituted or substituted more than once, such as, but not limited to, piperidinyl or quinolinyl groups, which are 2-, 3-, 4-, 5-, or 6- Attorney Docket No. 046483-7487W01(04056)

[0150] substituted, or disubstituted with groups such as those listed herein.

[0151] The term “hydrocarbon” or “hydrocarbyl” as used herein refers to a molecule or functional group that includes carbon and hydrogen atoms. The term can also refer to a molecule or functional group that normally includes both carbon and hydrogen atoms but wherein all the hydrogen atoms are substituted with other functional groups.

[0152] As used herein, the term “hydrocarbyl” refers to a functional group derived from a straight chain, branched, or cyclic hydrocarbon, and can be alkyl, alkenyl, alkynyk aryl, cycloalkyl, acyl, or any combination thereof. Hydrocarbyl groups can be shown as (Ca-Cb)hydrocarbyl, wherein a and b are integers and mean having any of a to b number of carbon atoms. For example, (Ci-C4)hydrocarbyl means the hydrocarbyl group can be methyl (Ci), ethyl (C2), propyl (C3). or butyl (C4), and (Co-Cb)hydrocarbyl means in certain embodiments there is no hydrocarbyl group.

[0153] The term “independently selected from” as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase “X1, X2, and X3are independently selected from noble gases” would include the scenario where, for example, X1, X2, and X3are all the same, where X1, X2, and X3are all different, where X1and X2are the same but X3is different, and other analogous permutations.

[0154] The term “ionizable lipid” as used herein refers to a lipid (e.g., a cationic lipid) or lipidoid having at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g, pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will be understood by one of ordinary' skill in the art that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or neutral lipid refers to the nature of the predominant species and does not require that all of the lipid be present in the charged or neutral form. Generally, ionizable lipids have a l<aof the protonatable group in the range of about 4 to about 7.

[0155] The term “local delivery.” as used herein, refers to delivery of an active agent or therapeutic agent such as a messenger RNA directly to a target site within an organism. For example, an agent can be locally delivered by direct injection into a disease site such as a tumor or other target site such as a site of inflammation or a target organ such as the liver, heart, pancreas, kidney, and the like.

[0156] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble Attorney Docket No. 046483-7487W01(04056)

[0157] in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids.” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

[0158] As used herein, “lipid encapsulated” can refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g, a protein cargo), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g, to form an SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle).

[0159] The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g, 1-1,000 nm) which includes one or more lipids and / or additional agents.

[0160] The term “lipid particle” is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g, mRNA), to a target site of interest. In the lipid particle of the disclosure, which is typically formed from a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle, the active agent or therapeutic agent may be encapsulated in the lipid, thereby protecting the agent from enzy matic degradation.

[0161] The term “monovalent” as used herein refers to a substituent connecting via a single bond to a substituted molecule. When a substituent is monovalent, such as, for example, F or Cl, it is bonded to the atom it is substituting by a single bond.

[0162] The term “mRNA” or “messenger RNA” as used herein refers to a ribonucleic acid sequences which encodes a peptide or protein. In certain embodiments, the mRNA may comprise a “transcript” that is produced by using a DNA template and encodes a peptide or protein. Typically, mRNA comprises 5’-UTR, protein coding region and 3 -UTR. mRNA can be produced by in vitro transcription from a DNA template. Methods of in vitro transcription are known to those of skill in the art. For example, various in vitro transfer kits are commercially available. According to the present invention, mRNA can be modified by further stabilizing modifications and cap formation in addition to the modifications according to the invention.

[0163] The term “neutral lipid” or “helper lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At phy siological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols. Attorney Docket No. 046483-7487W01(04056)

[0164] The term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0165] The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors (Pl. PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non -naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem, 260:2605-2608 (1985); Rossolini et al., Mai. Cell. Probes, 8:91-98 (1994)).

[0166] “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and py rimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0167] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly Attorney Docket No. 046483-7487W01(04056)

[0168] indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem, 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0169] The terms “patient.” “subject,” or “individual” are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In a non-limiting embodiment, the patient, subject or individual is a human.

[0170] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non- toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0171] As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids or bases, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

[0172] Suitable pharmaceutically acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of inorganic acids include hydrochloric, hydrobromic, hydriodic. nitric, carbonic, sulfuric (including sulfate and hydrogen sulfate), and phosphoric acids (including hydrogen phosphate and dihydrogen phosphate). Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic, carboxylic and sulfonic classes of organic acids, examples of which include formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, malonic, saccharin, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, trifluoromethanesulfonic, 2-hydroxyethanesulfonic, p-toluenesulfonic. sulfanilic, cyclohexylaminosulfonic, stearic, alginic, (3-hydroxybutyric, salicylic, galactaric and galacturonic acid.

[0173] Suitable pharmaceutically acceptable base addition salts of compounds described herein include, for example, ammonium salts, metallic salts including alkali metal, alkaline earth metal and transition metal salts such as, for example, calcium, magnesium, potassium, sodium and zinc salts. Pharmaceutically acceptable base addition salts also include organic salts made from basic amines such as, for example, N, N’ -dibenzyl ethylene-diamine, Attorney Docket No. 046483-7487W01(04056)

[0174] chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared from the corresponding compound by reacting, for example, the appropriate acid or base with the compound.

[0175] As used herein, the term “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid fdler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound described herein within or to the patient such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound(s) described herein, and not injurious to the patient. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound(s) described herein, and are physiologically acceptable to the patient. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound(s) described herein. Other additional ingredients that may be included in the pharmaceutical compositions used with the methods or compounds described herein are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0176] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein, and refer to a compound comprised of amino acid residues covalently linked by peptide Attorney Docket No. 046483-7487W01(04056)

[0177] bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. ‘"Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0178] The terms “polymer-conjugated lipid” and “conjugated lipid” are used interchangeably herein to refer to a lipid which is conjugated to one or more polymeric groups, which inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to diacylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (e.g., U. S. Pat. No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

[0179] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity’ does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical Attorney Docket No. 046483-7487W01(04056)

[0180] species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled ‘"A” and the antibody, will reduce the amount of labeled A bound to the antibody.

[0181] The term “substantially” as used herein refers to a majority of, or mostly, as in at least about 50%. 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term “substantially free of’ as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5. 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term “substantially free of’ can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4. 3.5, 3, 2.5, 2, 1.5, 1, 0.9. 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%. The term “does not substantially,” when used in conjunction with a process or event indicates that the process or event does not occur or occurs in a trivial amount, such that the process or event occurs about 0% or less than about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0. 3.5, 4.0, 4.5, or about 5.0% of the maximal amount that the process or event could occur.

[0182] The term “substituted” as used herein in conjunction with a molecule or an organic group as defined herein refers to the state in which one or more hydrogen atoms contained therein are replaced by one or more non-hydrogen atoms. The term “functional group” or “substituent” as used herein refers to a group that can be or is substituted onto a molecule or onto an organic group. Examples of substituents or functional groups include, but are not limited to, a halogen (e.g., F, Cl, Br, and I); an oxygen atom in groups such as hydroxy groups, alkoxy groups, aryloxy groups, aralkyloxy groups, oxo(carbonyl) groups, carboxyl groups including carboxylic acids, carboxylates, and carboxylate esters; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfoxide groups, sulfone groups, sulfonyl groups, and sulfonamide groups; a nitrogen atom in groups such as amines, hydroxyamines, nitriles, nitro groups, N-oxides, hydrazides, azides, and enamines; and other heteroatoms in various other groups. Non-limiting examples of substituents that can be bonded to a substituted carbon (or other) atom include F, Cl, Br, I, OR. OC(O)N(R)2, CN, NO, NO2, ONO2, azido, CF?, OCF3, R, O (oxo), S (thiono), C(O), S(O), methylenedioxy. Attorney Docket No. 046483-7487W01(04056)

[0183] ethylenedioxy, N(R)2, SR, SOR, SO2R, SO2N(R)2, SO3R, C(O)R, C(O)C(O)R, C(O)CH2C(O)R. C(S)R, C(O)OR. OC(O)R. C(O)N(R)2, OC(O)N(R)2, C(S)N(R)2. (CH2)O-2N(R)C(O)R, (CH2)O-2N(R)N(R)2, N(R)N(R)C(O)R, N(R)N(R)C(O)OR, N(R)N(R)CON(R)2, N(R)SO2R, N(R)SO2N(R)2, N(R)C(O)OR, N(R)C(O)R, N(R)C(S)R, N(R)C(O)N(R)2, N(R)C(S)N(R)2, N(COR)COR, N(OR)R, C(=NH)N(R)2, C(O)N(OR)R, and C(=NOR)R, wherein R can be hydrogen or a carbon-based moiety; for example. R can be hydrogen, (Ci-C100) hydrocarbyl, alkyl, acyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, or heteroarylalkyl; or wherein two R groups bonded to a nitrogen atom or to adjacent nitrogen atoms can together with the nitrogen atom or atoms form a heterocyclyl.

[0184] A “therapeutic"’ treatment is a treatment administered to a subject who exhibits signs of pathology, for the purpose of diminishing or eliminating those signs.

[0185] The term “therapeutic cargo” as used herein refers to any molecule (e.g., small molecules and macromolecules) or compound that provides a therapeutic or functional benefit to the targeted cell or tissue when delivered.

[0186] The term “therapeutic protein” as used herein refers to a protein or peptide which has a positive or advantageous effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In some embodiments, a therapeutic protein or peptide has curative or palliative properties and may be administered to ameliorate, relieve, alleviate, reverse, delay onset of or lessen the severity of one or more symptoms of a disease or disorder. A therapeutic protein or peptide may have prophylactic properties and may be used to delay the onset of a disease or to lessen the severity of such disease or pathological condition. The term “therapeutic protein” includes entire proteins or peptides, and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of a protein. Exemplar)’ therapeutic proteins include, but are not limited to, an analgesic protein, an anti-inflammatory protein, an anti -proliferative protein, an proapoptotic protein, an anti-angiogenic protein, a cytotoxic protein, a cytostatic protein, a cytokine, a chemokine, a grow th factor, a wound healing protein, a pharmaceutical protein, or a pro-drug activating protein. Therapeutic proteins may include growth factors (EGF, TGF-a, TGF- 0, TNF, HGF, IGF, and IL-1-8, inter alia) cytokines, paratopes, Fabs (fragments, antigen binding), and antibodies.

[0187] The terms “treat,” “treating” and “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject. Attorney Docket No. 046483-7487W01(04056)

[0188] Ionizable Lipid Compounds

[0189] In one aspect, the disclosure provides a compound of formula (I), or a salt, stereoisomer, or isotopologue thereof

[0190]

[0191] wherein:

[0192]

[0193] drug;

[0194] each occurrence of L1is independently selected from the group consisting of -O-, -N(RA)-, and -C(=O)-, and -(optionally substituted C1-C3 alkylenyl)-;

[0195] each occurrence of L2is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, -(optionally substituted C2-C8 heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-. -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C?-Cs cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, -(optionally substituted C2-C8 heteroarylenyl)-X-, and -X-;

[0196] each occurrence of X, if present, is independently selected from the group consisting of -N(R2c)-, -N(R3)-, -N(RA)-, -C(=O)-, and -O-;

[0197] R1is H;

[0198] R2a, R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of optionally substituted C1-C24 alkyl and optionally-substituted C1-C24 heteroalkyl;

[0199] each occurrence of R3is independently

[0200]

[0201] ;

[0202] each occurrence of L3is independently selected from the group consisting -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, - Attorney Docket No. 046483-7487W01(04056)

[0203] (optionally substituted Cs-Cs cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-Y-, -(optionally substituted C2-C12 alkenylenyl)-Y-, -(optionally substituted C1-C12 alkynylenyl)-Y-, -(optionally substituted C1-C12 heteroalkylenyl)-Y-, -(optionally substituted C3-C8 cycloalkylenyl)-Y-, -(optionally substituted C2-C8 heterocyloalkylenyl)-Y-, -(optionally substituted Ce-Cio ar lenyl)-Y-. -(optionally substituted C2-C8 heteroarylenyl)-Y-, and -Y-;

[0204] each occurrence of Y, if present, is independently selected from the group consisting of -N(RA)-, -O-, and -C(=O)-;

[0205] m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0206] n is 1, 2, 3, 4. 5, 6, 7. 8, 9, or 10;

[0207] o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0208] each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alky l; and

[0209] * indicates a bond between a C(=O) carbon and a nitrogen atom of

[0210]

[0211] In certain embodiments, L1is -(CH2)-. In certain embodiments, L1is -C(=O)-. In certain embodiments, L1is -O-.

[0212] In certain embodiments, -(L1)™- is -C(=O)O(CH2)2-.

[0213] In certain embodiments, L2is -(CH2)2-4-. In certain embodiments. L2is -N(CH3)-. In certain embodiments, L2is -N[(CH2)2-3N(R2d)(R2e)]-. In certain embodiments, L2is -C(=O)-.

[0214] - -N / l- - In certain embodiments, L2is -NH-. In certain embodiments, L2is \ /

[0215] In certain embodiments, the compound of formula (I) is:

[0216]

[0217] certain embodiments, the compound of formula (I) is: Attorney Docket No. 046483-7487W01(04056)

[0218]

[0219] certain embodiments, the compound of formula (I) is:

[0220]

[0221] certain embodiments, the compound of formula (I) is:

[0222]

[0223] (le). In certain embodiments, the

[0224]

[0225] compound of formula (I) is: (If). In certain embodiments, the compound of formula (I) is

[0226]

[0227] In certain embodiments, R2ais -(CH2)CH(OH)(optionally substituted C1-C22 alkyl). In certain embodiments, R2bis -(CH2)CH(OH)(optionally substituted C1-C22 alkyl). In certain embodiments, R2cis -(CH2)CH(OH)(optionally substituted C1-C22 alkyl). In certain embodiments, R2dis -(CH2)CH(OH)(optionally substituted C1-C22 alkyl). In certain embodiments, R2eis -(CH2)CH(OH)(optionally substituted C1-C22 alkyl).

[0228] OH

[0229] In certain embodiments, R2ais

[0230]

[0231] . In certain embodiments, R2ais

[0232] OH

[0233]

[0234] . In certain embodiments, R2ais

[0235]

[0236] In certain embodiments, R2ais Attorney Docket No. 046483-7487W01(04056)

[0237] OH OH

[0238]

[0239] 8. In certain embodiments, R2ais9. In certain embodiments. R2ais OH OH

[0240]

[0241] . In certain embodiments, R2ais

[0242]

[0243] . In certain embodiments, R2ais OH OH

[0244]

[0245] 12. In certain embodiments, R2ais13. In certain embodiments, R2bis OH OH X. CH3

[0246]

[0247] . In certain embodiments, R2bis6. In certain embodiments, R2bis OH OH

[0248]

[0249] 7. In certain embodiments, R2bis8. In certain embodiments, R2bis OH OH

[0250]

[0251] . In certain embodiments, R2bis

[0252]

[0253] In certain embodiments, R2bis OH OH CH3CH3

[0254] 11. In certain embodiments, R2bis12. In certain embodiments, R2bis OH OH

[0255] > CH3

[0256] 13. In certain embodiments, R2cis

[0257]

[0258] . In certain embodiments, R2cis OH OH

[0259]

[0260] 6. In certain embodiments, R2cis7. In certain embodiments, R2cis OH OH CH3X CH3

[0261] 8. In certain embodiments, R2cis9. In certain embodiments. R2cis OH

[0262]

[0263] . In certain embodiments, R2cis

[0264]

[0265] . In certain embodiments, R2cis OH OH

[0266]

[0267] 12. In certain embodiments, R2cis ' '13 In certain embodiments, R2dis OH OH

[0268]

[0269] 5. In certain embodiments, R2dis6. In certain embodiments, R2dis Attorney Docket No. 046483-7487W01(04056)

[0270] OH OH

[0271]

[0272] 7. In certain embodiments, R2dis8. In certain embodiments. R2dis OH OH

[0273]

[0274] . In certain embodiments, R2dis

[0275]

[0276] . In certain embodiments, R2dis OH OH

[0277]

[0278] 11. In certain embodiments, R2dis12. In certain embodiments, R2dis OH OH

[0279]

[0280] 13. In certain embodiments, R2eis5. In certain embodiments, R2eis OH OH

[0281] > CH3

[0282] 6. In certain embodiments, R2eis

[0283]

[0284] . In certain embodiments, R2eis OH OH

[0285]

[0286] . In certain embodiments, R2eis

[0287]

[0288] . In certain embodiments, R2eis OH OH

[0289] > CH3X CH3

[0290] 10. In certain embodiments, R2eis11. In certain embodiments, R2eis

[0291]

[0292] ,

[0293] In certain embodiments, the small molecule drug comprises at least one primary or secondary amine. In certain embodiments, the small molecule drug is useful for the treatment of cancer. In certain embodiments, the small molecule drug is an indoleamine 2,3-dioxygenase (IDO) inhibitor. In certain embodiments, the IDO inhibitor is indoximod (1-methy l-Z>-try ptophan).

[0294] In certain embodiments,

[0295]

[0296] In certain embodiments, the compound of formula (I) is selected from the group consisting of 110-SS-U-C10, 110-SS-11-C12, 110-SS-11-C14, L2A-SS-AA-C10, L2A-SS-AA-C12. L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14. DAB- SS-AA-C10. DAB-SS-AA-C12. DAB-SS-AA-C14. P2A-SS-AA-C10. P2A-SS-AA-C12, P2A-SS-AA-C14, 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14, G0-SS-AA-C10, Attorney Docket No. 046483-7487W01(04056)

[0297] G0-SS-AA-C12, and G0-SS-AA-C14.

[0298] In certain embodiments, each occurrence of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted cycloalkyl, optionally substituted heteroalkyl, optionally substituted heteroalkenyl, optionally substituted heterocycloalkyl, optionally substituted alkylenyl, optionally substituted alkenylenyl, optionally substituted alkynylenyl, optionally substituted heteroalkylenyl, optionally substituted cycloalkylenyl, optionally substituted heterocycloalkylenyl, optionally substituted arylenyl, and optionally substituted heteroarylenyl is independently optionally substituted with at least one substituent selected from the group consisting of Ci-Ce alkyd, Cs-Cs cycloalkyl, Ci-Ce haloalkyl, Ci-Cs haloalkoxy, phenoxy, halogen, CN, NCh, OH, N(R')(R”), C(=O)R’, C(=O)OR’, OC(=O)OR’. C(=O)N(R')(R ”), S(=O)2N(R )(R”), N(R’)C(=O)R”, N(R )S(=O)2R”, C2-Cs heteroaryl, and phenyl optionally substituted with at least one halogen, wherein each occurrence of R’ and R” is independently selected from the group consisting of H, Ci-Cs alky l, Cs-Cs cy cloalky l, Ci-Ce haloalkyl, benzyl, and pheny l, or wherein R’ and R” can combine with the nitrogen atom to which they are bound to form a C2-Cs heterocycloalkyl (e.g, cyclic tertiary' amine).

[0299] Ionizable Lipids and / or Cationic Lipids or Lipidoids

[0300] The scope of ionizable lipids contemplated for use in the present disclosure is not limited to ionizable lipidoids of formula (I). In the lipid nanoparticles of the disclosure, the cationic lipid or ionizable lipid may comprise, e.g., one or more of the following:

[0301] (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLinMC3DMA), [(4-hy droxybutyl)azanediyl] di(hexane-6, 1 -diyl) bis(2 -hexyldecanoate) (ALC-0315). heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl] amino} octanoate (SM-102), 1, 1 '-[[2-[4-[2-[[2-[bis(2-hy droxy dodecyl)amino] ethyl] (2-hy droxy dodecyl)amino] ethyl]- 1 -piperazinyl]ethyl]imino]bis-2-dodecanol (Cl 2-200), l,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N. N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3- 45 dimethylaminopropyl)- l,3]-dioxolane (D Lin-K-C3-D MA), 2, 2-dilinoleyl-4-(4-dimethylaminobutyl)-[l,3]-di oxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminomethyl-[l,3]-dioxane (DLin-K6-DMA), 2,2-dilinoleyl-4-N-methylpepiazino-[l,3]-dioxolane (DLin-K-MPZ). 2,2-dili-noleyl-4-dimethylaminomethyl-[1,3] -dioxolane (DLin-KDMA), l,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (D Attorney Docket No. 046483-7487W01(04056)

[0302] Lin-C-DAP), l,2-dilinoleyoxy-3-(dimethylaminoacetoxypropane (DLin-DAC), 1-2dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3 -trimethylaminopropane chloride salt (DLin-TMA. Cl), l,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP. Cl), l,2-dilinoleyloxy-3-(N-methylpiperazino)propane (D Lin-MPZ), 3-(N, N-dilinoleylamino)-l,2-propanediol (D LinAP), 3-(N, N-dioleylamino)-l,2-propanedio (DOAP), l,2-dilinoleyloxo-3-(2-N, N-dimethylamino)ethoxypropane (D Lin-EG-D MA), N, N-dioleyl-N, N-dimethylanrmonium chloride (DODAC), l,2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N, N-dimethylaminopropane (DSD MA). N-(l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-distearyl-N, N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTAP), 3-(N-(N’, N’dimethylaminoethane)-carbamoyl)cholesterol (DC -Choi), N-(l,2-dimyristyloxyprop-3-yl)-N, N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2 (spermine-carboxamidoethyl]-N. N-dimethy 1-1-propanamimumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5’-(cholest-5-en-3-beta-oxy)-3’-oxapentoxy)-3-dimethyl-l-(cis,cis-9’,l-2'-octadecadienoxy) propane (CpLinDMA), N, N-dimethyl-3,4-dioleyloxy benzylamine (DMOBA), 1.2-N, N’dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), l,2-N, N’-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2’"), or mixtures thereof. The ionizable lipids are not limited to those recited herein, and can further include ionizable lipids known to those skilled in the art, or described in PCT Application No. PCT / US2020 / 056255 and / or PCT Application No. PCT / US2020 / 056252, the disclosures of which are herein incorporated by reference in its entirety.

[0303] The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U. S. Application Publication No. US 2011 / 0256175, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S-DMA, DLm-2-DMAP, DLin-TMA. Cl, DLin-TAP. Cl, DLin-MPZ, DLinAP, DOAP. and DLin-EG-DMA. as well as additional cationic lipids, is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is herein Attorney Docket No. 046483-7487W01(04056)

[0304] incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U. S. Patent Publication No.

[0305] 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0306] Non-cationic Lipid

[0307] In the nucleic acid-lipid particles of the present disclosure, the non-cationic lipid may comprise, e.g., one or more anionic lipids and / or neutral lipids. In some embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

[0308] Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2’ -hydroxy ethyl ether, ch o les ten 1-4’-hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is known to one skilled in the art and described in U. S. Patent Nos. 8.058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378, all of which are hereby incorporated herein in their entireties for all purposes.

[0309] Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), ioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC). palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyloleyolphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate DOPE-mal), dipalmitoy lphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidylethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

[0310] Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids can be. for example, acyl groups derived from fatty acids having C10-C24 carbon chains, e.g, lauroyl, myristoyl, palmitoyl, stearoyl, or Attorney Docket No. 046483-7487W01(04056)

[0311] oleoyl. Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof such as cholestanol, cholestanone, cholestenone, coprostanol. cholesteryl-2 ’-hydroxy ethyl ether, cholesteryl-4’-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof.

[0312] Polymer-Conjugated Lipid(s)

[0313] In the nucleic acid-lipid particles of the present disclosure, the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEG) lipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic-polymer-lipid conjugates (CPLs), or mixtures thereof. In some embodiments, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.

[0314] PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5.000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEGOH), monomethoxypolyethylene glycolsuccinate (MePEGS), monomethoxypolyethylene glycolsuccinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycolamine (MePEG-NEb), monomethoxypolyethylene glycoltresylate (MePEG-TRES). and monomethoxypolyethylene glycolimidazolylcarbonyl (MePEG-IM). Other PEGs such as those described in U. S. Patent Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethyleneglycolacetic acid (MePEG-CEbCOOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.

[0315] In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. The conjugated lipid that inhibits aggregation of particles may comprise a PEG-hpid including, e g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEGDAA conjugate may be PEG-dilauryloxypropyl (C12), a PEG-dimyristyloxypropyl (C14), aPEG-dipalmity loxy propyl (Cie), a PEG-disteary 1 oxy propyl (Cis), or mixtures thereof.

[0316] Additional PEG-lipid conjugates suitable for use in the disclosure include, but are not limited to, mPEG2000-l,2-diO-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The Attorney Docket No. 046483-7487W01(04056)

[0317] synthesis of PEG-C-DOMG is described in PCT Application No. PCT / US08 / 88676, filed December 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, l-[8’-(l,2-dimyristoyl-3-propanoxy)-carboxamido-3’,6’-dioxaoctanyl] carbamoyl-methyl-poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U. S. Patent No. 7,404,969. the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0318] The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g, from about 1,000 daltons to about 5,000 daltons, from about 1.500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In some embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

[0319] In addition to the foregoing, it will be readily apparent to those of skill in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and poly dimethylacrylamide, polylactic acid, polygly colic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

[0320] In addition to the foregoing components, the particles (e.g, LNP) of the present disclosure can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (e.g, Chen et al., Bioconj. Chem., 11:433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in the present disclosure, and methods of making and using SPLPs and SPLP-CPLs, are disclosed, e.g, in U. S. Patent No. 6,852,334 and PCT Publication No. WO 00 / 62813, the disclosures of which are herein incorporated by reference in their entirety' for all purposes.

[0321] In certain instances, the conjugated lipid that inhibits aggregation of particles (e.g, PEG-lipid conjugate) may comprise from about 0.1 mol% to about 2 mol%, from about 0.5 mol% to about 2 mol%, from about 1 mol% to about 2 mol%, from about 0.6 mol% to about 1.9 mol%, from about 0.7 mol% to about 1.8 mol%, from about 0.8 mol% to about 1.7 mol%, from about 1 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.8 mol%, from about 1.2 mol% to about 1.7 mol%, from about 1.3 mol% to about 1.6 mol%, from about 1.4 mol% to about 1.5 mol%. or about 1. 1.1, 1.2, 1.3, 1.4. 1.5, 1.6, 1.7, 1.8. 1.9, or 2 mol% (or any fraction thereof or range therein) of the total lipid present in the particle. Attorney Docket No. 046483-7487W01(04056)

[0322] In the lipid nanoparticles of the present disclosure, the active agent or therapeutic agent may be fully encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation. In some embodiments, a nucleic acid-lipid particle comprising a nucleic acid such as a messenger RNA (i.e., mRNA) is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37° C. for at least about 30, 45, or 60 minutes or at least about 2, 3, 4, 5, 6, 7, 8. 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34. or 36 hours. In other embodiments, the active agent or therapeutic agent (e.g., nucleic acid such as siRNA) is complexed with the lipid portion of the particle. One of the benefits of the formulations of the present disclosure is that the lipid particle compositions are substantially non-toxic to mammals such as humans.

[0323] Lipid Nanoparticle (LNP) Compositions

[0324] In another aspect, the disclosure provides a lipid nanoparticle (LNP) composition. In certain embodiments, the LNP comprises at least one ionizable lipid. In certain embodiments, the at least one ionizable lipid comprises or consists essentially of the compound of formula (I). In certain embodiments, the LNP comprises at least one neutral lipid. In certain embodiments, the LNP comprises at least one cholesterol lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises at least one polymer-conjugated lipid and / or a modified derivative thereof. In certain embodiments, the LNP comprises at least one nucleic acid encoding an immunomodulatory protein. In certain embodiments, the LNP comprises at least one small molecule anticancer agent. In certain embodiments, the small molecule anticancer agent is attached to at least one component of the LNP by a cleavable linker.

[0325] In certain embodiments, the immunomodulatory protein is interleukin 12 (IL- 12) and the small molecule anticancer agent is indoximod.

[0326] In certain embodiments, the at least one ionizable lipid compound comprises less than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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, or 90 mol% of the LNP. In certain embodiments, the at least one Attorney Docket No. 046483-7487W01(04056)

[0327] ionizable lipid compound comprises about 10. 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. 36. 37. 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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, or 90 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22. 23.24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35. 36. 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47. 48. 49. 50. 51. 52, 53, 54, 55, 56, 57, 58, 59, 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, or 90 mol% of the LNP.

[0328] In certain embodiments, the at least one ionizable lipid compound comprises less than about 50 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises about 50 mol% of the LNP. In certain embodiments, the at least one ionizable lipid compound comprises greater than about 50 mol% of the LNP.

[0329] In certain embodiments, the at least ionizable lipid compound comprises or consists essentially of at least one ionizable lipid selected from the group consisting of 110-SS-l 1-C10, 110-SS-l 1-C12, 110-SS-l 1-C14, L2A-SS-AA-C10. L2A-SS-AA-C12, L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14, DAB-SS-AA-C10, DAB-SS-AA-C12, DAB-SS-AA-C14, P2A-SS-AA-C10, P2A-SS-AA-C12, P2A-SS-AA-C14, 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14, G0-SS-AA-C10, G0-SS-AA-C12, and GO-SS-AA-CI4.

[0330] In certain embodiments, the at least one neutral lipid comprises less than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 1, 2. 3, 4, 5, 6, 7, 8, 9. 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or about 40 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39. or about 40 mol% of the LNP.

[0331] In certain embodiments, the at least one neutral lipid comprises less than about 10 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises about 10 mol% of the LNP. In certain embodiments, the at least one neutral lipid comprises greater than about 10 mol% of the LNP.

[0332] In certain embodiments, the neutral lipid comprises or consists essentially of at least one neutral lipid selected from the group consisting of dioleoylphosphatidylethanolamine Attorney Docket No. 046483-7487W01(04056)

[0333] (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC). In certain embodiments, the neutral lipid comprises or consists essentially of dioleoylphosphatidylethanolamine (DOPE).

[0334] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises less than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49. 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61. 62. 63. 64, 65, 66, 67, 68, 69, 70, 71, 72, 73. 74. or about 75 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55. 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or about 75 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises greater than about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or about 75 mol% of the LNP.

[0335] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises less than about 38.5 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises about 38.5 mol% of the LNP. In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises greater than about 38.5 mol% of the LNP.

[0336] In certain embodiments, the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol.

[0337] In certain embodiments, the at least one polymer-conjugated lipid comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1. 1.2, 1.3, 1.4, 1.5. 1.6, 1.7, 1.8, 1.9. 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7. 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5. 9.6, 9.7, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5. 10.6. 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3. 11.4. 11.5. 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5, 14.6, 14.7, 14.8, 14.9, or about 15 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises about 0.1, 0.2, 0.3, 0.4. 0.5, 0.6, 0.7, 0.8. 0.9, 1.0, 1.1, 1.2. 1.3, 1.4, 1.5, 1.6. 1.7, 1.8, 1.9, 2.0. 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, Attorney Docket No. 046483-7487W01(04056)

[0338] 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7. 6.8, 6.9, 7.0, 7.1. 7.2, 7.3, 7.4, 7.5. 7.6, 7.7, 7.8, 7.9. 8.0, 8.1, 8.2, 8.3. 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4, 14.5. 14.6, 14.7, 14.8, 14.9, or about 15 mol% of the LNP. In certain embodiments, the at least one polymer-conjugated lipid comprises greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 06, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0. 6.1, 6.2, 6.3, 6.4, 6.5. 6.6, 6.7. 6.8, 6.9. 7.0, 7.1, 7.2, 7.3. 7.4, 7.5, 7.6, 7.7. 7.8, 7.9, 8.0, 8.1. 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, 14.0, 14.1, 14.2, 14.3, 14.4. 14.5. 14.6, 14.7, 14.8, 14.9, or about 15 mol% of the LNP.

[0339] In certain embodiments, the at least one polymer-conjugated lipid comprises less than about 1.5 mol%. In certain embodiments, the at least one polymer-conjugated lipid comprises about 1.5 mol%. In certain embodiments, the at least one polymer-conjugated lipid comprises greater than about 1.5 mol%.

[0340] In certain embodiments, the at least one polymer-conjugated lipid comprises or consists essentially of l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).

[0341] In certain embodiments, the LNP has a molar ratio of (a): (b): (c): (d) of about 50:10:38.5:1.5.

[0342] In certain embodiments, the at least one nucleic acid encoding an immunomodulatory protein comprises RNA or DNA. In certain embodiments, the at least one nucleic acid encoding an immunomodulatory protein comprises mRNA. In certain embodiments, the mRNA encodes an interleukin. In certain embodiments, the interleukin is interleukin 12 (IL-12).

[0343] Methods

[0344] In another aspect, the disclosure provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject. In certain embodiments, the method comprises Attorney Docket No. 046483-7487W01(04056)

[0345] administering to the subject at least one LNP of the disclosure or the pharmaceutical composition of the disclosure.

[0346] In certain embodiments, the disease or disorder is at least one selected from the group consisting of cancer, an immune disease or disorder, and a metabolic disease or disorder.

[0347] In certain embodiments, the disease or disorder is cancer.

[0348] In certain embodiments, the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer.

[0349] Pharmaceutical Compositions

[0350] In another aspect, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticle (LNP) of the present disclosure and at least one pharmaceutically acceptable earner. In certain embodiments, the composition further comprises at least one adjuvant. In certain embodiments, the composition is a vaccine.

[0351] Such a pharmaceutical composition may consist of at least one composition of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0352] In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng / kg / day and 100 mg / kg / day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng / kg / day and 1,000 mg / kg / day.

[0353] The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w / w) active ingredient. Attorney Docket No. 046483-7487W01(04056)

[0354] Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

[0355] In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

[0356] The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the t pe and age of the veterinary or human patient being treated, and the like.

[0357] The formulations of the pharmaceutical compositions described herein may be prepared by any method know n or hereafter developed in the art of pharmacology7and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory7ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

[0358] As used herein, a “unit dose” is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g, about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0359] Although the descriptions of pharmaceutical compositions provided herein are Attorney Docket No. 046483-7487W01(04056)

[0360] principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0361] In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier.

[0362] Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN*), solubilized gelatins (e., GELOFUSINE4), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington's Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0363] The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0364] Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, Attorney Docket No. 046483-7487W01(04056)

[0365] parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and / or fragrance-conferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, "additional ingredients” include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

[0366] The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total w eight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzy l alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

[0367] The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary' range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by w eight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total w eight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelflife of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

[0368] Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, w ater, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or w etting agents, emulsifying agents, demulcents, preservatives, buffers, salts, Attorney Docket No. 046483-7487W01(04056)

[0369] flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to. sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxy cetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or w -propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

[0370] Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily " liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil. fractionated vegetable oils, and mineral oils such as liquid paraffin.

[0371] A pharmaceutical composition of the invention may7also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for Attorney Docket No. 046483-7487W01(04056)

[0372] example, sweetening or flavoring agents.

[0373] Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (i. e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.

[0374] Administration / Dosing

[0375] The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0376] Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (z.e., composition) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition; the duration of the treatment; other drugs, compounds or matenals used in combination with the composition; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic composition of the disclosure is from about 0.01 mg / kg to 100 mg / kg of body weight / per day of active agent (i.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition without undue experimentation. Attorney Docket No. 046483-7487W01(04056)

[0377] The composition may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day. once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition dosed per day may be administered, in non-limiting examples, every day, every other day, every' 2 days, every' 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.

[0378] Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0379] A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0380] In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity' of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary’ dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such a therapeutic composition for the treatment of a disease or disorder in a patient.

[0381] In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a Attorney Docket No. 046483-7487W01(04056)

[0382] week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

[0383] The amount of active agent of the composition(s) of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 p g to about 6.000 mg, about 100 p g to about 5,500 mg, about 200 p g to about 5,000 mg, about 400 p g to about 4.000 mg, about 800 p g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

[0384] In some embodiments, the dose of active agent (z. e., nucleic acid) present in the composition of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg. or less than about 10 mg, or less than about 5 mg. or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

[0385] In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce Attorney Docket No. 046483-7487W01(04056)

[0386] one or more symptoms of a disease or disorder in a patient.

[0387] The term “container” includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product. However, it should be understood that the instructions may contain information pertaining to the compound’s abi 1 i ty to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

[0388] Administration

[0389] Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g, sublingual, lingual, (trans)buccal, (trans )urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0390] Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

[0391] Parenteral Administration

[0392] As used herein, “parenteral administration” of a pharmaceutical composition includes Attorney Docket No. 046483-7487W01(04056)

[0393] any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrastemal injection, and kidney dialytic infusion techniques.

[0394] Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PC A) devices. Formulations for parenteral administration include, but are not limited to. suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to. suspending, stabilizing, or dispersing agents. In certain embodiments of a formulation for parenteral administration, the active ingredient is provided in dry (z.e., powder or granular) form for reconstitution with a suitable vehicle (e.g, sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0395] The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a nontoxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di -glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically Attorney Docket No. 046483-7487W01(04056)

[0396] acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0397] EXAMPLES

[0398] Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

[0399] Materials and Methods

[0400] Materials and antibodies

[0401] DLin-MC3-DMA was obtained from MedChem Express (Monmouth Junction. New Jersey, USA). l,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG 2000) were purchased from Avanti Polar Lipids. 2,2'-disulfanediylbis(ethan-l-ol), 1-methyl-D-tryptophan, hexane- 1,6-diol, and polyamines heads were purchased from Sigama Aldrich (Burlington, Massachusetts, USA). Epoxy decane (CIO), epoxy dodecane (Cl 2), epoxytetradecane (Cl 4), and 4-nitrophenyl carbonochloridate were purchased from Tokyo Chemical Industry' (TCI). All of Organic solvents were purchased from Fisher Scientific. Firefly luciferase (FLuc) mRNA(5moU) (L-7202) was purchased from TriLink Bio Technologies. Anti-mouse CD3-PE (clone: 17A2, catalog number: 12-0032-82), anti-mouse CD4-FITC (clone: GK1.5, catalog number: 11-0041-82), anti-mouse CD8-APC (clone: 5H10, catalog number: MCD0805) were purchased from ThermoFisher. Anti-mouse CD44-BV421 (clone: IM7, catalog number: 103039), antimouse CD62L-BV605 (clone: MEL-14, catalog number: 104437), anti-mouse CD279 (PD- l)-BV510 (clone: 29F.1A12, catalog number: 135241), anti-mouse FOXP3-BV421 (clone: MF-14, catalog number: 126419), and anti-mouse CD25-BV605 (clone: PC61, catalog number: 102035) were purchased from BioLegend.

[0402] Preparation of lipid nanoparticles

[0403] For the preparation of MC3 LNPs, DLin-MC3-DMA, DSPC, cholesterol, DMG-PEG were mixed at a molar ratio of 50 / 10 / 38.5 / 1.5 and dissolved in 200 proof ethanol. For the preparation of other LNPs, ionizable lipid, DOPE, cholesterol, DMG-PEG were mixed at a molar ratio of 50 / 10 / 38.5 / 1.5 and dissolved in ethanol. mRNA was dissolved in 10 mM citrate buffer (pH=3). For in vitro screening experiments, the ethanol phase and the citrate Attorney Docket No. 046483-7487W01(04056)

[0404] buffer phase were mixed using pipette mixing. For other in vitro experiments and animal experiments, the ethanol phase and the citrate buffer phase were mixed in a microfluidic chip device at a flow rate ratio of 1:3. LNPs were then purified by dialyzing against 1 x PBS (dialysis cassette MWCO 20kDa) for 2 h to remove ethanol and citrate buffer. A 0.22 pM pore size filtration device was used to remove any aggregates and the as obtained LNPs were stored at 4 °C fridge until use. A Quant-iT RiboGreen RNA assay was used to quantify the mRNA encapsulation efficiency. mRNA encoding firefly luciferase was used to formulate the LNPs for in vivo screening experiments.

[0405] In vitro screening of pLNPs

[0406] HepG2 cells were seeded in 96-well plates at a density of 5000 per well in 100 uL cell culture medium (DMEM, 10% FBS, 1% P / S) overnight. Then fresh DMEM medium was changed and pLNPs were added in the wells at a dose of 62.5 ng FLuc per well (MC3 LNPs severed as a control). The expression of FLuc mRNA and cell viability were measured after treating cells for 24 h by Luciferase Reporter 1000 Assay System (#E4550. Promega, Madison, Wisconsin) and cell viability was measured using a CellTiter-Glo Luminescent Cell Viability Assay (#G7572, Promega) according to manufacturer’s protocols.

[0407] In vivo screening of pLNPs

[0408] Female mice were i.v. injected FLuc mRNA loaded pLNPs at a dose of 2.5 pg per mouse (MC3 LNPs served as a positive control). D-luciferin potassium salt (150 mg / kg) was intraperitoneally injected into mice 6 h post-injection and bioluminescence imaging was performed using an in vivo imaging system (PerkinElmer). For in vivo screening based on tumor model, pLNPs encapsulated FLuc mRNA were intratumorally injected at a dose of 2.5 pg per mouse. After 6 h, mice were intraperitoneally injected with D-luciferin potassium salt (150 mg / kg). After that, the FLuc mRNA expression was measured by bioluminescence imaging using an in vivo imaging system (PerkinElmer).

[0409] IVIS imaging

[0410] A PerkinElmer Lumina III IVIS was used to collect the IVIS images shown in this study. Images were collected under the defined luminescence channel using the auto exposure mode. Living Image 4.5 software and the Auto ROI function were used to quantify the luminescence level in each image. Attorney Docket No. 046483-7487W01(04056)

[0411] Labeling LNPs with DiO

[0412] 1 mL LNPs was added to a 1.5 mL tube with 2 pL DMSO containing DiO (1 mg / mL). After vortex mixing and incubation at room temperature (25 min), the mixture was loaded on a centrifugal filter device (MWCO: 10 kDa) and LNP-DiR / DiO was washed with PBS three times to remove free DiO. The purified LNP-DiO nanoparticles w ere stored at 4 °C fridge before use.

[0413] ELISA for IL-12 characterization in vitro

[0414] MC38 cells were incubated in 12 well plate at a density of 5 x 105cells per well in 1 mL DMEM overnight. Then PBS, MC3 IL-12 LN, G0-6C-AA-C12 IL-12 LNPs, and GO-SS-AA-C12 IL-12 pLNP were added at a dose of 0.5 pg / mL IL-12 mRNA per well (PBS served as negative control). After 16 h co-culture, the supernatants of each group were collected. And MC38 cells were lysed by using 300 pL lysis buffer. The protein level of IL- 12 in each group was measured by ELISA kit from Thermo Fisher scientific (catalogue number: 88-7121-22), following the manufacture's instruction.

[0415] Immunofluorescence for analyzing intracellular IL-12

[0416] MC38 cells were seed in confocal imaging dishes at a density7of 5 x 104cells per dish in 300 pL DMEM overnight. Then MC3 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP, and GO-SS-AA-C12 IL- 12 pLNP were added at a dose of 0.5 pg / mL IL- 12 mRNA. respectively. PBS was also added as negative control. After 16 h, cells were fixed by fixation buffer (catalogue number: 420801, BioLegend) and treated by intracellular staining permeabilization w ash buffer (catalogue number: 421002, BioLegend) for 10 min. After that, cells w ere treated with anti-mouse IL-12 antibody at a concentration of 5 pg / mL in PBST buffer containing 5% BSA at 37 °C for 3 h. Then, cells were washed with PBS for 2 times, stained by IL-12 (p40 / p70) rat-anti mouse PE antibody (catalogue number: BDB562038, Thermo Fisher Scientific) at 37 °C for 1 h. Subsequently, cells were wash with PBS for 3 time, Hoechst (10 pg / mL) was added for staining cell nucleus at 37 °C for 15 min. Afterwards, cells were washed with PBS for 3 times and 300 uL PBS was added for following confocal imaging.

[0417] Flow cytometry analysis for intracellular IL- 12 characterization

[0418] MC38 cells were seed in 6 well plate at a cell density of 3 x 105in 1.5 mL DMEM overnight. MC3 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP, and G0-SS-AA-C12 IL-12 pLNP were treated with MC38 cells at a dose of 0.5 pg / mL IL-12 mRNA per well at 37 °C for 16 h. Attorney Docket No. 046483-7487W01(04056)

[0419] Fixation buffer was used to fix cells for 15 min. Then cells were treated by intracellular staining permeabilization wash buffer for 10 min. Anti-mouse IL- 12 PE antibody in permeabilization wash buffer was added and incubated for 45 min. After that, cells were collected by centrifuging at 400 g for 5 min. Then MC38 cells were washed with PBS for 3 times and subjected for flow cy tometry7analysis.

[0420] In vitro indoximod (1-methyl-D-tryptophan) release from G0-SS-AA-C12 pLNP Typically, G0-SS-AA-C12 pLNP with the concentration of prodrug ionizable lipid at 1.7 mg / mL were dispersed in 100 pL PBS buffer containing 5 mM GSH. Afterwards, the mixture solution was placed in a well of 96-well microdialysis plate (catalog number:

[0421] A50472, Thermo Fisher Scientific) and then immersed into 1 mL PBS containing 5 mM GSH at 37 °C. Then 50 pL external PBS buffer solution was taken for HPLC analysis at detected wavelength of 286 nm at different time point. The calibration curve of indoximod was used to quantify the drug release.

[0422] G0-SS-AA-C12 pLNP -medicated increase ofT cell cytokines (TNF-a. IFN-y, and granzyme B) secretion in vitro

[0423] MC38 cells were incubated with either G0-SS-AA-C12 IL-12 pLNP or

[0424] G0-SS-AA-C12 FLuc pLNP at a mRNA dose of 0.5 pg / mL per well for 24 h, after which the culture supernatants were collected. Primary mouse CD3* T cells were isolated from the spleens of female C57BL / 6 mice using a CD3* T cell isolation kit (catalog number: 480024, BioLegend) and subsequently activated with mouse T cell activation beads (catalog number: 11456D, Thermo Fisher) in complete T cell culture medium lacking mouse IL-2 (mIL-2) for 3 days. After removal of the activation beads, the CD3+T cells were resuspended in medium. Supernatants from the treated MC38 cells were then mixed with T cell culture medium (without mIL-2) at a volume ratio of 1 / 1 and used to culture tine CD3 T cells for 48 h (PBS served as a negative control). Following this incubation, the T cells were harvested and stimulated with a cell stimulation cocktail (catalog number; 00-4970-93, Thermo Fisher) and a protein transport inhibitor cocktail (catalog number: 00-4930-93, Thermo Fisher) for 4 h. After stimulation, the cells were collected and stained for surface markers on ice for I h. They were then washed with PBS buffer, fixation and permeabilization buffers, respectively. The intracellular cytokines were then stained with corresponding antibodies. Finally, the cells were subjected to flow cytometry to assess intracellular TNF-a, IFN-y, and granzy me B production. Attorney Docket No. 046483-7487W01(04056)

[0425] GO-SS-AA-C 12 pLNP -mediated T cell proliferation in vitro

[0426] MC38 cells were treated with G0-SS-AA-C12 IL-12 pLNP and G0-SS-AA-C12 FLuc pLNP for 24 h at a dose of 0.5 pg / mL per well, respectively. Then, supernatants were collected and mixed with T cell culture-medium at a volume ratio of 1 / 1. Primary CD3+T cells were isolated from spleens of female C57BL / 6 mice and activated by T cell activation beads for 3 days. After removal of all activation beads, T cells were dyed with CFSE cell proliferation kit (catalog number: C34554, Thermo Fisher) and further cultured with T cell culture medium (without mIL-2) for 3 days. After, CD3 T cells were harvested and subjected for flow cytometry to analyzed T cell proliferation.

[0427] GO-SS-AA-C 12 pLNP -mediated suppression of T cell exhaustion in vitro

[0428] To investigate the effects of G0-SS-AA-C12 pLNP on T cell exhaustion, MC38-OVA cancer cells were co-cultured with primary OT-I CD •• T cells isolated from the spleens of OT-I female mice using a CD31T cell isolation kit (catalog number: 480024, BioLegend). The cells were plated at an effector-to-target (E: T) ratio of 1:1 6-well plate (MC38-OVA: 5 x 105cells; OT-I CD3+T cells: 5 x 103cells). GO-SS-AA-C 12 IL- 12 pLNP or G0-SS-AA-C12 FLuc pLNP was then added to the co-culture system at a mRNA dose of 05 pg / mL per well, followed by incubation for three days. Subsequently, an additional 5 x 105MC38-OVA cancer cells were added to the well along with a second administration of pLNP at the same mRNA dose (0.5 pg / mL), and the culture w as maintained for another three days This process w as repeated 4 times in total At the end of the experiment, all cells w ere harvested and analyzed by flow cytometry to assess T cell activation, as indicated by CD69 and CD25 expression, and to evaluate changes in PD- 1 levels, a key marker of T cell exhaustion.

[0429] Intratumor administration of LNPs to evaluate the effect of cancer immunotherapy

[0430] 6-8 weeks C57BL / 6 mice were subcutaneously inoculated ~1 x 106MC38 cells on day 0. The treatments were performed in tumor bearing mice at the indicated time points (day 6,8,10) by intratumoral injection of GO-SS-AA-C 12 FLuc pLNP, G0-6C-AA-CI2 IL- 12 LNP, and G0-SS-AA-C12 IL-12 pLNP at a dose of 2.5 pg mRNA per mouse, respectively. Control mice were injected intratumorally with 50 uL of PBS. Then tumors were measured by a precision caliper. At the same time, the body weights of mice were recorded.

[0431] RNA-sequencing Attorney Docket No. 046483-7487W01(04056)

[0432] MC38 tumor tissues were harvested on day 12 after three intratumor injection of G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP. G0-SS-AA-C12 IL-12 pLNP, and PBS, respectively. After that, the total RNA was extracted. Sequencing was performed in Novogen Co. and the differentially expressed genes were analyzed using DEGSeq R package (1.20.0). The -values were adjusted using the Benjamini & Hochberg method. The threshold of corrected / J- value of 0.05 and log2 (Fold change) of 1 was selected to determine the significantly differential expressed genes.

[0433] Characterization

[0434] ’H-NMR were recorded using a Bruker 400 MHz NMR spectrometer. LC-MS was performed on a Waters Acquity LCMS system equipped with UV-Vis and MS detectors. The hydrodynamic size, poly dispersity index (PDI) and zeta potential of LNPs were measured using a Malvern Zetasizer Nano ZS90. The morphology7of LNPs was characterized by a cryo-electron microscope (Titan Krios, Thermo Fisher). The mRNA encapsulation efficiency and the pKa of LNP were determined using a modified Quant-iT RiboGreen RNA assay (Invitrogen, Carlsbad, California, USA) and a 6-(ptoluidinyl) naphthalene-2-sulfonic acid (TNS) assay, respectively.

[0435] Cell lines and animals

[0436] The HepG2, and MC38 cell lines were originally obtained from ATCC. All cells were tested mycoplasma negative before use. 6-8 weeks female C57BL / 6 mice were purchased from Jackson laboratory. All mice were housed in a specific-pathogen-free animal facility at ambient temperature, air humidity 40%-70% and 12-h dark / 12-h light cycle.

[0437] mRNA synthesis

[0438] Codon optimized firefly Luciferase sequence, mouse IL-12 p70 sequence were cloned into a mRNA production plasmid. The plasmid was transcribed in vitro in the presence of 1-methyl pseudouridine modified nucleoside, co-transcriptionally capped using the CleanCapTM technology (TriLink), and purified with cellulose to remove double-stranded RNAs. Purified rnRNAs were precipitated in ethanol, washed with ethanol and were resuspended in nuclease-free water. Electrophoresis, dot blot, endotoxin assays were performed for quality7control. All rnRNAs were stored in a -80 °C freezer until use.

[0439] Flow cytometry Attorney Docket No. 046483-7487W01(04056)

[0440] Mice were perfused with 50 mL PBS from the left ventricle to remove blood. Spleens, or tumor tissues were isolated and were cut into 3x3x3 mm cubes. After enzymatic digestion with collagenase V (1 mg / mL), RNase (40 U / mL), and DNase I (40 U / mL) for 30 min at 37 °C, tissues were ground on a 70 pm cell strainer to obtain single-cell suspensions. The cells were washed three times. Then cellular surface staining was performed for 30 min on ice with antibodies. After that, cells washed with PBS and analyzed using flow cytometry (BD LSR II).

[0441] Measuring pKa of mRNA-LNP

[0442] TNS reagent was dissolved in ultrapure water at a concentration of 0.16 mM. Then, buffered solutions of 150 mM sodium chloride, 20 mM sodium phosphate, 25 mM ammonium citrate, and 20 mM ammonium acetate were created and adjusted to different pH values ranging from 2.5 to 9.5 in increments of 0.5. After that, 2.5 pL of the LNP was added to each pH-adjusted buffer solution in a black- wall black-bottom 96-well plate (three wells for each pH). TNS reagent was then added to each well so that the final TNS concentration was 6 pM. The 96-well plate was incubated in a dark location for 5 min. Fluorescence at an excitation / emission of 322 nm / 431 nm was measured using a fluorescence plate reader. The data was fitted to a sigmoidal curve. and the pKa was calculated as the pH corresponding to half of the maximum TNS fluorescence value.

[0443] Statistics

[0444] Statistical analyses were performed using GraphPad Prism 7.0 software. The error bars indicate the mean ±standard deviation (s.d.). To assess statistical differences between two groups, the Student’s t-test was employed. For evaluating statistical differences among multiple grounds, a one-way ANOVA with Tukey’s post hoc test was utilized. In animal survival experiments, the P values were determined using the log-rank test based on the Kaplan-Meier method.

[0445] Example 1: Compounds synthesis

[0446] Synthesis of Nitro-SS-Nitro

[0447] 4-Nitrophenyl carbonochloridate (2.4 equiv.), triethylamine (3 equiv.) and 50 mLTHF were charged into a reaction flask. Then 2-hydroxylethyl disulfide (1 equiv.) was dissolved in 50 mL THF and slowly added into flask. The mixture was stirred under room temperature overnight. Upon completion, the reaction mixture was filtered to remove undissolved Attorney Docket No. 046483-7487W01(04056)

[0448] substance and collect filtrates. After removing all the solvents on a rotary evaporator, the residues were further purified by CombiFlash Rf-200i on silica gel using hexane / ethyl acetate (v / v = 6 / 1) as eluent to afford Nitro-SS-Nitro (yield: 80%). 'H-NMR (400 MHz, CDCh, ppm): 8 8.30 (d, J= 9.2 Hz, 4H), 7.41 (d, J= 9.2 Hz, 4H), 4.59 (t, J= 6.5 Hz, 4H), 3.11 (t, J = 6.5 Hz, 4H).

[0449] Synthesis of 4-Nitro-SS-AA

[0450] Nitro-SS-Nitro (1.2 equiv.) and triethylamine (1.5 equiv.) were dissolved in 20 mL THF in a reaction flask and stirred at 0 °C. Then 1 -methyl-D-tryptophan (1 equiv.) was dissolved in a mixture of THF and NaHCOs aqueous (v / v = 1 / 3) and slowly added into reaction flask. The reaction mixture was allowed to warm to room temperature under stirring for 3 h. Upon completion, THF was removed under reduced pressure. The residues were poured into 100 mL ethyl acetate and washed with 50 mL saturated NaCl solution three times. The organic layer was collected and dried over anhydrous Na2SOr, filtered and evaporated to dryness under reduced pressure. The residues were further purified by silica gel column chromatography using hexane / ethyl acetate = 1 / 1 as eluent to afford 4-Nitro-SS-AA (yield: 52%). 1HNMR (400 MHz, DMSO-d6, ppm): 8 8.32 (d, J= 9.2 Hz, 2H), 7.59-7.47 (m, 4H), 7.36 (s, 1H), 7.17-7.10 (m, 2H), 7.07-6.97 (m, 1H), 4.47 (t, J= 6.1 Hz, 1H), 4.27-4.08 (m, 3H), 3.72 (s, 3H). 3.20-2.92 (m, 6H). LC-MS (m / z): Calcd for [M+Na]+: 586.09. found: 585.80; Calcd for [M+H]+: 564.11, found: 563.82.

[0451] Synthesis of prodrug amine heads

[0452] Amine head (3 equiv.). triethylamine (1.5 equiv.) and 10 mL THF were charged into reaction flask and stirred at 0 °C. Then 4-Nitro-SS-AA (1 equiv.) dissolved in THF slowly added into the flask. After stirring for 2 h, all solvents were removed by reduced pressure. The residues were further purified by preparative HPLC to afford prodrug amine heads for the following synthesis. 1H NMR (400 MHz, DMSO-de, ppm) for G0-SS-AA: 3 7.50 (d. J= 7.8 Hz, 1H), 7.32 (d, J= 8.2 Hz, 1H), 7.07 (d, J= 7.5 Hz, 1H), 6.96 (t, J = 7.3 Hz, 1H), 4.31 (dd, J= 16.3, 3.9 Hz, -1H), 4.14-4.02 (m, 3H), 3.68 (s, 3H), 3.53-3.47 (m, 19H), 3.01-3.00 (m, 2H), 2.99-2.97 (m, 4H), 2.86 (s, 6H), 2.55 (s, 8H), 2.42 (s, 4H). LC-MS (m / z): Calcd for [M+H]+: 941.47, found: 941.48: Calcd for [M+2H]2+: 471.24, found: 471.44; Calcd for [M+3H]3+: 314.50, found: 314.74. Attorney Docket No. 046483-7487W01(04056)

[0453] Synthesis of prodrug ionizable lipids

[0454] Prodrug ionizable lipids were synthesized by an S 2 reactions between different polyamine lipid cores and epoxide-terminated lipid tails. Taking synthesis of G0-SS-AA-C12 as example, G0-SS-AA(l equiv.) and 1,2-epoxy dodecane (7.2 equiv.) were dissolved in ethanol in a glass vail. The reaction mixture was stirred at 80 °C for 2 days. Then solvents were removed to afford crude product for the in vitro and in vivo screening for FLuc mRNA delivery. The top performance prodrug ionizable lipid G0-SS-AA-C12 was purified by CombiFlash Rf-200i. G0-SS-AA-C12. 1H NMR (400 MHz, DMSO-t / 6): 57.51 (dd, J= 19.0, 7.4 Hz, 1H), 7.41-7.21 (m, 1H), 7.20-6.73 (m, 3H), 4.38-4.09 (m, 5H), 3.72 (s, 3H), 3.66-3.54 (m. 6H), 3.50-3.45 (m. 16H), 3.15 (s, 9H). 3.05 (s, 2H), 2.88 (t, J= 5.3 Hz. 6H), 2.70-2.60 (m, 14H), 2.58 (d, J = 6.2 Hz, 10H), 2.41 (s, 3H), 1.44-1.35 (m, 13H), 1.23 (s, 99H), 0.86-0.84 (m, 21H).

[0455] Synthesis ofNitro-6C-Nitro

[0456] 4-Nitrophenyl carbonochloridate (2.4 equiv.), triethylamine (3 equiv.) and 50 mLTHF were charged into a reaction flask. Then 1,6-hexanediol (1 equiv.) was dissolved in 50 mL THF and slowly added into flask. The mixture was stirred under room temperature overnight. Upon completion, the reaction mixture was filtered to remove undissolved substance and collect filtrates. After removing all the solvents on a rotary evaporator, the residues were further purified by combiflash on silica gel using hexane / ethyl acetate (v / v = 6 / 1) as eluent to afford Nitro-6C-Nitro (yield: 72%). 1H NMR (400 MHz, CDCh, ppm): 8 8.34-8.26 (m, 4H), 7.44-7.37 (m, 4H), 4.33 (d, J= 6.5 Hz, 4H), 1.83 (p, J= 6.7 Hz, 4H), 1.61-1.44 (m, 4H).

[0457] Synthesis of 4-Nitro-6C-AA

[0458] Nitro-6C -Nitro (1.2 equiv.) and triethylamine (1.5 equiv.) were dissolved in 20 mL THF in a reaction flask and stirred at 0 °C. Then 1 -methyl-D-tryptophan (1 equiv.) was dissolved in a mixture of THF and NaHCCh aqueous (v / v = 1 / 3) and slowly added into reaction flask. The reaction mixture was allowed to warm to room temperature under stirring for 3 h. Upon completion, THF was removed under reduced pressure. The residues were poured into 100 mL ethyl acetate and washed with 50 mL saturated NaCl solution three times. The organic layer was collected and dried over anhydrous Na2SO4, filtered and evaporated to dryness under reduced pressure. The residues were further purified by silica gel column chromatography using hexane / ethyl acetate = 1 / 1 as eluent to afford 4-Nitro-SS-AA Attorney Docket No. 046483-7487W01(04056)

[0459] (yield: 48%). LC-MS (m / z): Calcd for [M+Na]+: 550.18, found: 549.90; Calcd for [M+H]+: 528.20. found: 527.64.

[0460] Synthesis of G0-6C-AA

[0461] GO (3 equiv.), triethylamine (1.5 equiv.) and 10 mL THF were charged into reaction flask and stirred at 0 °C. Then 4-Nitro-6C-AA (1 equiv.) dissolved in THF slowly added into the flask. After stirring for 2 h, all solvents were removed by reduced pressure. The residues were further purified by preparative HPLC to afford G0-6C-AA for the following synthesis.

[0462] 1H NMR (400 MHz, DMSO-d6, ppm): 5 7.51 (d, J= 8.0 Hz, 1H), 7.28 (s, 1H), 6.97 (d, J = 19.1 Hz, 3H), 3.93-3.89 (m, 5H), 3.70 (s, 3H). 3.62 (dq. J= 8.4, 2.5 Hz, 19H), 2.79 (s, 7H), 2.52 (s, 8H), 2.33 (s, 6H), 1.41 (s, 4H), 1.22 (s, 2H), 1.16 (s, 2H). LC-MS (m / z): Calcd for [M+H]+: 905.56, found: 905.75; Calcd for [M+2H]2+: 453.29, found: 453.60; Calcd for [M+3H]3+: 302.52, found: 302.79.

[0463] Synthesis of G0-6C-AA-C12

[0464] G0-6C-AA(l equiv.) and 1,2-epoxy dodecane (7.2 equiv.) were dissolved in ethanol in a glass vail. The reaction mixture was stirred at 80 °C for 2 days. Then solvents were removed to afford crude product for the in vitro and in vivo screening for FLuc mRNA delivery. The G0-6C-AA-C12 was purified by CombiFlash Rf- 200i. G0-6C-AA-C12. 1H NMR (400 MHz, DMSO-d6, ppm): 5 7.23 (d, J= 2.4 Hz, 1H), 7.10-7.05 (m, 3H), 3.92 (s, 5H), 3.70 (s, 3H), 3.63 (dd, J= 10.8, 5.8 Hz, 10H), 3.42 (s, 8H), 2.92-2.90 (m, 6H), 2.68 (d, J = 2.0 Hz, 13H), 2.64-2.62 (m, 8H), 2.34 (s, 11H), 1.68-1.08 (m. 118H), 0.81 (d, J= 11.6 Hz, 20H).

[0465] Example 2: Rational design and synthesis of pIL libraries for in vitro and in vivo screening

[0466] Conventional ILs were synthesized via an SN2 reaction between polyamine heads and epoxide-terminated lipid tails. To synthesize an indoximod pIL, an intermediate prodrug structure, 4-Nitro-SS-AA, was first generated. The hydroxyls of 2-hydroxyethyl disulfide were first protected by 4-nitrophenyl carbonochloridate to generate reactive carbonates for a subsequent reaction with indoximod (1-methyl-D-try ptophan) to obtain 4-Nitro-SS-AA (FIG.

[0467] 7). Presence of a disulfide linkage in this structure enables intracellular release of indoximod via GSH cleavage. Next, 4-Nitro-SS-AA was reacted with seven amine heads to generate Attorney Docket No. 046483-7487W01(04056)

[0468] seven prodrug poly amine heads (FIG. 1 A and FIG. 8). These structures were then reacted with one of three epoxide-terminated alkyl tails for 48 h at 80°C to yield a library of 21 pILs (FIG. 9). These pILs are denoted by a specific nomenclature: 110 to GO refers to the seven amine head groups, SS refers to the presence of a disulfide linkage, AA refers to the presence of I -methyl -D-tr tophan. and C10 / C12 / C14 refers to the length of the alkyl tails in the ionizable lipid. For example. ‘“G0-SS-AA-C12” has a GO amine head, a disulfide linkage, 1-methyl-£>-try ptophan, and C12 alkyl tails.

[0469] Next, the crude pILs were combined with l,2-dioleoyl-snglycero-3-phosphoethanolamine (DOPE), l,2-dimyristoylrac-glycero-3-methoxypolyethylene gly col-2000 (DMG-PEG), and cholesterol in ethanol and pipette-mixed with 1 -methylpseudouridine (ml\| / )-modified FLuc mRNAto formulate 21 pLNPs. The molar ratio of pIL / cholesterol / DOPE / DMG-PEG was kept constant at 50 / 38.5 / 10 / 1.5 (FIG. 2A). The pLNP library was screened in HepG2 cells, an immortalized human hepatocellular carcinoma cell line. In brief, HepG2 cells were treated with pLNPs at a dose of 62.5 ng / well of mRNA. After 8 h, luciferase substrate was added, and luminescence intensity was quantified via plate reader. FDA-approved DLin-MC3-DMA (MC3) LNPs were used as a positive control.

[0470] Among the 21 pILs, GO-based pILs had the highest transfection efficacy in vitro among the seven amine head groups (FIG. 10). The top eight pILs were advanced for study of in vivo efficacy following intravenous (i.v.) administration of pLNPs encapsulating FLuc mRNA at a dose of 2.5 pg / mouse. After 6 h. in vivo luciferase expression was quantified via bioluminescence imaging. All pLNPs primarily transfected the liver with G0-SS-AA-C12 pLNP facilitating 4-fold greater hepatic transfection than MC3 LNPs (FIG. 11).

[0471] Next, it was verified that pILs could be used to transfect an additional cancer cell line. A similar workflow was repeated, this time using MC38 cells, a mouse colon adenocarcinoma cell line, in vitro and a MC38 subcutaneous tumor mouse model (FIG. 2A). Treatment of MC38 cells in vitro with the library of pLNPs encapsulating FLuc mRNA showed that G0-SS-AA-C12 pLNP had the highest transfection efficacy (FIG. 2B). All pLNPs exhibited low cytotoxicity in vitro (FIG. 12). Additionally, to emphasize the structural advantage of the pIL, another control lipid, termed G0-6C-AA-C12, was also designed that could not release drug by substituting the disulfide linkage with carbon-carbon linkage (FIG.

[0472] 11). Interestingly, G0-SS-AA-C12 pLNP facilitated 1.5-fold greater transfection of MC38 cells than LNPs formulated with a structural analog that lacked the disulfide linkage (G0-6C-AA-C12) (FIG. 14). Since G0-6C-AA-C12 has a carbon-carbon bond instead of a disulfide linkage, it is unable to release indoximod in the cytoplasm. This implies that the prodrug Attorney Docket No. 046483-7487W01(04056)

[0473] itself within the pIL aids in pLNP -mediated mRNA transfection.

[0474] Given the interest in applying pLNPs for the treatment of solid tumors, the efficacy of the top nine pILs was subsequently evaluated in vivo following MC38 intra-tumoral injection of pLNPs encapsulating FLuc mRNA at a dose of 2.5 pg / mouse. After 6 h, total luminescence intensity7within the MC38 flank tumor was quantified, demonstrating that G0-SS-AA-C12 pLNP not only outperformed the transfection efficacy of other pLNPs in the library7but also FDA-approved MC3 LNP (FIGs. 2C-2D).

[0475] Then, G0-SS-AA-C12 and its uncleavable structural analog G0-6C-AA-C12 were purified and their structures were verified. Purified G0-SS-AA-C12, G0-6C-AA-C12, or MC3 ionizable lipid were combined with the standard excipients and microfluidic-mixed with FLuc mRNA to generate anew set of LNPs. G0-SS-AA-C12 pLNP and G0-6C-AA-C12 LNP were characterized for their physiochemical properties. Transmission electron microscopy (TEM) visualized uniform, multilamellar, complex spherical morphologies of both G0-SS-AA-C12 pLNP and G0-6C-AA-C12 LNP with hydrodynamic diameters of 43 nm and 78 nm. poly dispersity indices of 0.12 and 0.26, and slightly positive surface charge (FIGs. 15-16). The FLuc mRNA encapsulation efficiency (EE) of G0-SS-AA-C12 pLNP and G0-6C-AA-C12 LNP was determined to be 91.2% and 89.6%, respectively; and their pKa values were approximately 5.82 and 6.06, respectively (FIGs. 15-17). Next, the cellular uptake of G0-SS-AA-C12 pLNP, G0-6C-AA-C12, and MC3 LNPs were compared via 3,3'-dioctadecyloxacarbocyanine perchlorate (DiO) labeling. Confocal imaging showed greater intracellular uptake of G0-SS-AA-C 12 pLNP relative to MC3 and G0-6C-AA-C12 LNPs in MC38 cells (FIG. 2E and FIG. 18). Moreover, the endosomal escape of lead pLNP w as also assessed. A higher percentage of G0-SS-AA-C12 pLNP escaped endosomes in comparison to MC3 LNP in MC38 cells (FIG. 2F), demonstrating a potential mechanism for enhanced efficacy for the lead pLNP.

[0476] Example 3: G0-SS-AA-C12 pLNP facilitate efficient IL-12 mRNA expression and resistance to T cell exhaustion in vitro

[0477] Encouraged by the strong in vitro and in vivo efficacy of G0-SS-AA-C12 pLNP, the potential of this platform to deliver IL-12 mRNA was next assessed. G0-SS-AA-C 12, G0-6C-AA-C12, and MC3 LNPs encapsulating IL- 12 mRNA w ere formulated and MC38 cells were treated with these formulations at a dose of 0.5 pg / mL of mRNA. After 16 h, both the supernatant and cells were harvested for ELISA-based analysis of extracellular and intracellular IL-12 protein expression, respectively (FIG. 3A). Although both extracellular Attorney Docket No. 046483-7487W01(04056)

[0478] and intracellular IL-12 protein was detected in all three treatment groups, G0-SS-AA-C12 pLNP facilitated 44-fold greater extracellular IL- 12 protein and 8.6-fold greater intracellular IL-12 protein than MC3 LNP (FIGs. 3B-3C). Moreover, treatment with G0-SS-AA-C12 pLNP resulted in 2.8-fold greater extracellular IL-12 protein and 4.4-fold greater intracellular IL-12 protein than G0-6C-AA-C12 LNP (FIGs. 3B-3C). Flow cytometry and confocal imaging also confirmed strong intracellular IL-12 expression in MC38 cells after GO-SS-AA-CI2 pLNP treatment (FIGs. 3D-3G).

[0479] The drug release capability of G0-SS-AA-C12 pLNP was then further investigated. Specifically, G0-SS-AA-C12 pLNP dispersion (1 x PBS) with GSH at a concentration of 5 mM which corresponds to the concentration of GSH in cancer cell was placed in a microdialysis plate with immersion of 1.0 mL PBS medium containing 5 mM GSH. The disulfide linkages of G0-SS-AA-C12 were dissociated via GSH triggering and generating a mercapto group for the following intramolecular cyclic reaction to release indoximod (FIG.

[0480] 3H). The indoximod concentrations at different time points in the PBS was quantified by high-performance liquid chromatography (HPLC) (FIGs. 19-21). The results showed that -87% indoximod was released from G0-SS-AA-C12 pLNP after treatment with GSH for 72 h (FIG. 31). The potential of G0-SS-AA-C12 pLNP to limit T cell exhaustion was subsequently explored using an in vitro serial culture assay (FIG. 3 J). In brief, MC38 cells were treated with PBS (negative control) or G0-SS-AA-C12 pLNP encapsulating either IL-12 mRNA or FLuc mRNA. After 24 h, the supernatant of each group was acquired and used to culture primary mouse CD3+T cells. Following 24 h of culture, T cells w ere harvested and analyzed for PD-1 expression, a key marker ofT cell exhaustion, via flow cytometry'. T cells cultured in the media of G0-SS-AA-C12 pLNP-treated MC38 cells had significantly reduced PD-1 expression in comparison to the negative control group (FIGs. 3K-3L), regardless of mRNA cargo. Interestingly, G0-SS-AA-C12 pLNP encapsulating IL-12 mRNA mediated greater suppression of PD-1 expression than those encapsulating FLuc mRNA (FIGs. 3K-3L). Taken together, these results imply a synergistic mechanism between the released indoximod (IDO inhibitor) and translated IL- 12 in resisting T cell exhaustion.

[0481] The capacity of G0-SS-AA-C12 pLNP to mitigate T cell exhaustion was evaluated by co-culturing primary OT-I CD3+T cells with ovalbumin-modified MC38 (MC38-OVA) cancer cells at a 1:1 effector-to-target (E: T) ratio. G0-SS-AA-C12 IL-12 pLNP and G0-SS-AA-C12 FLuc pLNP w ere added at 0.5 pg / well for three days. Cells treated with PBS served as a negative control. Every three days, equivalent numbers of MC38-OVA cells and corresponding amounts of mRNA-pLNP were added, for a total of five cycles, to mimic Attorney Docket No. 046483-7487W01(04056)

[0482] chronic T cell stimulation within the TME. After the final stimulation, cells were collected and analyzed via flow cytometry for CD69 and CD25 expression, markers of T cell activation, and for PD-1 expression, a marker of T cell exhaustion (FIG. 3M). Treatment with G0-SS-AA-C12 IL-12 pLNP upregulated CD69 and CD25 on OT-I CD3+T cells (FIGS. 3N-30) and downrcgulated PD-1 (FIG. 3P) relative to control groups. Interestingly, GO-SS-AA-C12 pLNP encapsulating IL-12 mRNAmediated greater suppression of PD-1 expression than pLNP encapsulating FLuc mRNA (FIG. 30). Together, these results imply a synergistic mechanism between the released indoximod and translated IL- 12 in resisting T cell exhaustion.

[0483] Example 4: Evaluation of G0-SS-AA-C12 pLNP encapsulating IL-12 mRNA in vivo for cancer immunotherapy in MC38 tumor mouse model

[0484] Given the efficacy of G0-SS-AA-C12 pLNP in vitro in priming T cells towards an effector phenotype and in vivo in facilitating intra-tumoral mRNA delivery, the therapeutic efficacy of this platform was evaluated in an MC38 tumor mouse model. To highlight the synergistic effect of the lead mRNA-pLNP platform, composed of both a small molecule IDO inhibitor and therapeutic IL- 12 mRNA, two control LNP formulations were prepared. GO-SS-AA-C12 pLNP encapsulating FLuc mRNA was used to assess the effect of IDO inhibitor only, while G0-6C-AA-C12 pLNP encapsulating IL-12 mRNA was used to evaluate the antitumor efficacy of IL- 12 protein only (FIG. 22). After subcutaneous injection of MC38 cells into the right flank of mice, G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP, or G0-SS-AA-C12 IL-12 pLNP at an mRNA dose of 2.5 pg / mouse were administered intratumorally on day 6, day 8, and day 10 (FIG. 4A). PBS treatment via the same procedure was used as a negative control. G0-SS-AA-C12 pLNP encapsulating IL- 12 mRNA greatly suppressed tumor growth (FIG. 4B), clearing all tumors within 30 days of model initiation (FIGs. 4B-4C). In comparison, G0-SS-AA-C12 pLNP encapsulating FLuc mRNA and G0-6C-AA-C 12 pLNP encapsulating IL- 12 mRNA only partially inhibited tumor growth (FIGs.

[0485] 4B-4C), demonstrating the utility of this synergistic strategy. All pLNPs were well-tolerated with no effect on body weight (FIG. 4D) or major organ (z.e., heart, liver, spleen, lung, and / or kidney) histology (FIG. 23) relative to PBS-treated mice. Finally, in terms of survival, PBS-treated mice succumbed to tumor burden before day 45, G0-SS-AA-C12 FLuc pLNP and G0-6C-AA-C12 IL-12 LNP moderately increased survival, and all G0-SS-AA-C12 IL-12 pLNP-treated mice survived to the study endpoint (FIG. 4E).

[0486] To further characterize the importance of co-formulating the IDO inhibitor with IL-12 Attorney Docket No. 046483-7487W01(04056)

[0487] mRNAin a single LNP, three additional control groups were investigated: free IL- 12 mRNA, free IDO inhibitor, and G0-6C-AA-C12 IL- 12 LNP with free IDO inhibitor. G0-SS-AA-C12 IL- 12 pLNP demonstrated superior anti-tumor efficacy compared to these control groups. Neither free IL-12 mRNA nor free IDO inhibitor alone significantly suppressed tumor growth. G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor only partially inhibited tumor progression with mean tumor volumes rising rapidly after day 22. In contrast,

[0488] G0-SS-AA-CI2 IL- 12 pLNP completely suppressed tumor growth and nearly eradicated all tumors within 30 days. All mice in G0-SS-AA-C12 IL-12 pLNP -treated group survived to study endpoint. Splenic CD4+, CD8+, and regulatory' T cell response was also assessed for each of these groups on day 12. Notably, G0-SS-AA-C12 IL-12 pLNP elicited a robust CD8+T cell response and diminished the number of CD4+and regulatory T cells compared to control groups. Overall, these data confirmed that co-formulation of both an IDO inhibitor and IL- 12 mRNA in a single LNP was integral to induction of a potent immune response and anti-tumor efficacy.

[0489] Next was investigated the antitumor mechanism of G0-SS-AA-C12 IL-12 pLNP. Given that IL-12 is known to activate cells in both the innate and adaptive immune compartment and the spleen plays a major role in immunoregulation, the effect of pLNPs specifically on splenic T cell populations was explored. A separate cohort of mice was inoculated with MC38 tumors and treated via the same procedure and groups described previously. On day 12. spleens were harvested and analyzed via flow cytometry. GO-SS-AA-C12 IL-12 pLNP more effectively biased T cells in the spleen towards a CD8+effector phenot pe relative to PBS and other LNP-treated control groups (FIGs. 4F-4G). In addition, G0-SS-AA-C12 IL-12 pLNP induced a significant reduction in CD4+T cells in the spleen in comparison to control groups, likely due to elimination of regulatory T cells that can dampen the antitumor response (FIG. 4F and FIG. 4H). It was also observed that G0-SS-AA-C12 IL-12 pLNP elicited immune memory' development, specifically in effector memory (CD44 CD62L ) and central memory' (CD44+CD62L+) CD4+and CD8+T cell populations (FIGs. 4I-4L, FIG. 24, and FIGs. 25A-25B). Finally, G0-SS-AA-C12 IL-12 pLNP significantly reduced the presence of regulatory T cells (CD4 CD25 FOXP3 ) in the spleen (FIG. 4M and FIGs. 26-27). Taken together, these results demonstrate that G0-SS-AA-C12 IL- 12 pLNP achieves their antitumor efficacy through immune priming towards an effector T cell phenotype, while also generating long-term immune memory that may protect against tumor rechallenge. Attorney Docket No. 046483-7487W01(04056)

[0490] Example 5: G0-SS-AA-C12 IL-12 pLNP increases effector T cell infiltration and exerts immunostimulatory activity in the tumor microenvironment

[0491] Having characterized the impact of G0-SS-AA-C12 IL-12 pLNP on splenic immunoregulation, their influence on T cell infiltration was next evaluated within MC38 tumors. Again, the subcutaneous MC38 tumor mouse model was prepared and treated via the same procedure and groups described previously. On day 12, tumors were harvested and analyzed via flow cytometry (FIG. 5A). G0-SS-AA-C12 IL-12 pLNP significantly increased the presence of CD8+T cells within the TME (FIG. 5B) relative to control groups, likely due to IL-12-mediated recruitment and stimulation of the effector functions of T cells. Notably, G0-SS-AA-C12 IL-12 pLNP facilitated a greater increase in CD8+T cells than G0-6C-AA-C12 IL-12 LNP, recapitulating the in vitro results that demonstrated the role of the disulfide bond in improving mRNA-LNP transfection efficacy (FIG. 2B, FIG. 2D, and FIG. 5B). There was no significant difference between the number of CD4+cells between treatment groups (FIG. 5C). In terms of T cell exhaustion, G0-SS-AA-C12 FLuc pLNP (which only release IDO inhibitor) significantly downregulated PD-1 expression on CD4+and CD8+T cells in the TME compared to PBS-treated tumors (FIGs. 5D-5E. FIG. 28, and FIGs. 29A-29C). GO-SS-AA-C12 IL-12 pLNP further downregulated CD4+and CD8+T cell exhaustion in the TME relative to G0-SS-AA-C12 FLuc pLNP, demonstrating further synergy in therapeutic function between IL12 and the IDO inhibitor (FIGs. 5D-5E, FIG. 28, and FIGs. 29A-29C). Thus, G0-SS-AA-C12 IL-12 pLNP enhances effector T cell infiltration and reduce T cell exhaustion in the TME via synergistic mechanisms, likely leading to the complete tumor regression demonstrated in the MC38 tumor mouse model (FIGs. 4B-4C).

[0492] To understand the impact of the IDO inhibitor and IL-12 release from pLNPs on the gene expression profile of cells within the TME, bulk RNA sequencing (RNA-seq) was performed on harvested MC38 tumors (FIG. 5A). IL-12 exerts many of its downstream effects via interferon gamma (IFN-y). Thus, both GO-SS-AA-C 12 IL-12 pLNP and G0-6C-AA-C12 IL-12 LNP-treated tumors had an upregulation of IFN-y transcripts by 6.3-fold and 2.1 -fold, respectively, relative to PBS-treated tumors (FIG. 5F). In contrast, tumors treated with G0-SS-AA-C12 FLuc pLNP, which have no IL-12 mRNA cargo, had no significant upregulation of IFN-y. More broadly, GO-SS-AA-C 12 IL-12 pLNP shifted the gene expression profile of the TME from “cold” to “hot,” as evidenced by significant upregulation of pro-inflammatory cytokines, leukocyte chemokines, and T cell priming and dendritic cell (DC) activation genes relative to both GO-SS-AA-C 12 FLuc pLNP and G0-6C-AA-C12 IL-12 pLNP (FIG. 5G, FIGs. 30A-30B, FIGs. 31A-31D, and FIGs. 32A-32D). In particular, GO- Attorney Docket No. 046483-7487W01(04056)

[0493] SS-AA-C12 IL-12 pLNP induced the upregulation of multiple pro-inflammatory cytokine transcripts (e.g., Tnfsfll. Tnfsfl5, and III 5), likely contributing to its antitumor efficacy (FIG.

[0494] 5H). Moreover, consistent with increased T cell infiltration into the TME, G0-SS-AA-C12 IL-12 pLNP significantly upregulated the levels of Cxcl9 (3.7-fold), CxcllO (3.6-fold), and Cd3e (2.2-fold), which are vital mediators of T cell recruitment (FIG. 51). Increased levels of Cd80, Cd86, Cd28. Cd226, Irf4, and Ccr7 transcripts, known T cell priming genes, after G0-SS-AA-CI2 IL-12 pLNP treatment were consistent with the observed increase in activated effector T cells in the TME (FIG. 5J). Simultaneously, T-cell co-stimulatory molecule-related gene (Icos, 3.7-fold) (FIG. 5J), cytotoxic protein-related gene (Gzma, 2.7-fold) (FIG. 5M) and apoptosis-related genes (e g., Ifriy, Klrcl, Klrc4) (FIG. 5L) were also upregulated in G0-SS-AA-C12 IL- 12 pLNP -treated tumors, signifying enhanced cytotoxic T cell differentiation. Finally, an elevation oiSell (3.7-fold), a memory7T ceil marker, was observed in GO-SS-AA-C12 IL-12 pLNP-treated tumors (FIG. 5M), which is congruent with the observed induction of splenic memory T cell populations (FIGs. 4H-4M), Overall, these data imply that G0-SS-AA-C12 IL- 12 pLNP fundamentally modulate the TME by exerting immunostimulatory effects that leads to the antitumor efficacy of this platform.

[0495] Example 6: Evaluation of anti-tumor efficacy and systemic toxicity of G0-SS-AA-C12 IL-12 pLNP platform after intravenous administration

[0496] Given the robust anti-tumor efficacy of G0-SS-AA-CI2 IL-12 pLNP after intratumoral administration, the efficacy of pLNP after intravenous administration was explored in a murine MC38 tumor model. Mice were treated with free IL-12 mRNA, free IDO inhibitor, G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP, G0-6C-AA-C12 IL-12 LNP plus free IDO inhibitor, and G0-SS-AA-C12 IL-12 pLNP at a mRNA dose of 2.5 pg / mouse on day 6, day 8, and day 10 (FIG. 34A). Treatment with PBS served as a negative control. Treatment with G0-SS-AA-C12 IL-12 pLNP, G0-6C-AA-C12 IL-12 LNP, or G0-6C-AA-C12 IL-12 LNP with free IDO inhibitor inhibited tumor grow th (FIG. 34B). In comparison, free IL-12 mRNA and free IDO inhibitor failed to exhibit anti-tumor function relative to PBS group (FIG. 34C). Long-term monitoring of tumor progression and mice survival showed that while LNPs encapsulating IL-12 mRNA partially suppressed tumor burden, they w ere unable to eradicate tumors (FIG. 34D).

[0497] Next, systemic toxicity of the seven distinct treatment groups was evaluated. Serum was collected on day 12 for assessment of cytokine levels, liver function markers, and pro-inflammatory' cytokines (FIG. 35A). All LNPs encapsulating IL-12 mRNA elevated serum Attorney Docket No. 046483-7487W01(04056)

[0498] levels of IL-6, TNF-a, and IFN-y after intravenous injection (FIGs. 35B-35D), while treatment with free IL- 12 mRNA, free IDO inhibitor, and G0-SS-AA-C12 FLuc LNP resulted in serum cytokine levels comparable to the negative control (FIGs. 35B-35D). Similarly, all LNPs encapsulating IL-12 mRNA increased AST and ALT levels compared to PBS group (FIG. 36). In contrast, G0-SS-AA-C12 IL-12 pLNP administered via intratumoral injection did not increase serum IL-6 and TNF-a relative to PBS treatment (FIGs. 35E-35G), and these cytokine levels were 3.4-fold and 2.2-fold lower, respectively, than those after intravenous administration of pLNP (FIGs 35H-35I). No significant hepatotoxicity was observed after intratumoral injection of any treatment (FIG. 37). Together, although pLNP encapsulating IL-12 mRNA has moderate anti-tumor efficacy after intravenous injection, systemic toxicity limits this platform's potential via this route of administration. In contrast, intratumoral injection of pLNP encapsulating IL-12 mRNA boasts significant anti-cancer efficacy with minimal systemic toxicity.

[0499] Example 7: Local administration of G0-SS-AA-C12 IL-12 pLNP protects against tumor rechallenge and drives regression of distal tumors

[0500] Since G0-SS-AA-C12 IL- 12 pLNP elicited a substantial memory T cell response upon intra-tumoral administration, it was further tested whether this platform could be used to protect against tumor rechallenge. First, mice were inoculated with subcutaneous MC38 tumors (-100 mm3) and administered G0-SS-AA-C12 IL-12 pLNP intratumorally at an mRNA dose of 2.5 pg / mouse on day 6, day 8, and day 10. On day 30, MC38 cancer cells were injected into the flank of mice with primary MC38 tumors that were previously eliminated by pLNPs treatment or mice naive to prior treatment as a control (FIG. 6A). All mice that successfully cleared their primary tumors after G0-SS-AA-C12 IL-12 pLNP treatment rejected growth of a secondary tumor (FIGs. 6B-6C) and survived to the terminal endpoint (> 60 days) without detectable tumor burden (FIG. 6D). In contrast, control mice grew tumors (FIGs. 6B-6C) and all succumbed to their tumor burden within 40 days (FIG.

[0501] 6D). No loss in body weight was observed in the treatment group after tumor rechallenge (FIG. 6E). Taken together, G0-SS-AA-C12 IL-12 pLNP not only has the potential to treat and eliminate primary tumors, but it can also induce sufficient immune memory to protect against local tumor regrowth.

[0502] It was next probed whether the global immunomodulatory effect of G0-SS-AA-C12 IL-12 pLNP could drive the regression of tumors distal to the primary tumor injection site. To test this, mice were inoculated with two subcutaneous MC38 tumors, one on each flank. Attorney Docket No. 046483-7487W01(04056)

[0503] Then, only the right tumor was injected with either PBS (negative control), G0-SS-AA-C12 FLuc pLNP, G0-6C-AA-C12 IL-12 LNP, or G0-SS-AA-C12 IL-12 pLNP at an mRNA dose of 2.5 pg / mouse on day 6, day 8, and day 10 (FIG. 6F). Impressively, G0-SS-AA-C12 IL-12 pLNP induced complete regression of not only the treated tumor but also the contralateral tumor (FIGs. 6G-6H). As a result, all mice treated with G0-SS-AA-C12 IL-12 pLNP survived to the terminal endpoint, nearly 3 months after tumor inoculation (FIG. 61). In contrast, both flank tumors of G0-SS-AA-C12 FLuc pLNP-treated mice showed rapid growth and resulted in minimal survival benefit relative to PBS-treated mice, demonstrating that IDO inhibitor alone was not sufficient for overall therapeutic efficacy (FIGs. 6H-6I). For the G0-6C-AA-C 12 IL-12 pLNP-treated mice, the untreated distal tumors were unsuccessfully cleared by pLNPs without a cleavable IDO inhibitor (FIG. 6H) leading to limited long-term survival (FIG. 61). No body weight changes were observed in all treatment groups, demonstrating the biocompatibility of pLNP platforms (FIG. 6J). Finally, changes in T cell populations within the distal TME were explored via flow cytometry of harvested contralateral tumors. Akin to the perturbation of T cell populations within the primary’ tumor (FIG. 5B), treatment with GOSS- AA-C 12 IL- 12 pLNP resulted in greater CD8+T cells in the contralateral tumor relative to PBS-treated and G0-SS-AA-C12 FLuc pLNP-treated groups (FIGs. 6K-6L and FIG. 33). Of note, G0-SS-AA-C12 IL-12 pLNP induced a similar CD8+T cell response but had a much lower reduction of CD4+T cells in the contralateral tumor relative to G0-6C-AA-C12 IL-12 LNP (FIG. 6K-6L), likely resulting in ineffective tumor regression due to persistence of tumor-protective regulatory T cells. Taken together, these results imply that both IDO inhibitor release and IL-12 expression are necessary' within pLNPs for clearance of distal tumors and maximal therapeutic efficacy of this platform.

[0504] Enumerated Embodiments

[0505] The following exemplary' embodiments are provided, the numbering of w hich is not to be construed as designating levels of importance:

[0506] Embodiment 1 provides a compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:

[0507]

[0508] wherein: Attorney Docket No. 046483-7487W01(04056)

[0509]

[0510] e drug;

[0511] each occurrence of L1is independently selected from the group consisting of -O-, -N(RA)-, and -C(=O)-, and -(optionally substituted C1-C3 alkylenyl)-;

[0512] each occurrence of L2is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted Cs-Cs cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, -(optionally substituted C2-C8 heteroarylenyl)-X-, and -X-;

[0513] each occurrence of X, if present, is independently selected from the group consisting of -N(R2c)-, -N(R3)-, -N(RA)-, -C(=O)-, and -O-;

[0514] R1is H;

[0515] R2a, R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of optionally substituted C1-C24 alkyl and optionally substituted C1-C24 heteroalkyl;

[0516] R2d

[0517] - - {L3^-N^

[0518] each occurrence of R3is independently R2e;

[0519] each occurrence of L3is independently selected from the group consisting -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2-Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-Y-, -(optionally substituted C2-C12 alkenylenyl)-Y-, -(optionally substituted C1-C12 alkynylenyl)-Y-, -(optionally substituted C1-C12 heteroalkylenyl)-Y-, -(optionally substituted C3-C8 cycloalkylenyl)-Y-, -(optionally substituted C2-C8 heterocyloalkylenyl)-Y-, -(optionally substituted C6-C10 arylenyl)-Y-, - Attorney Docket No. 046483-7487W01(04056)

[0520] (optionally substituted C2-C8 heteroarylenyl)-Y-, and -Y-;

[0521] each occurrence of Y, if present, is independently selected from the group consisting of -N(RA)-, -O-, and -C(=O)-;

[0522] m is consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0523] n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0524] o is 1, 2, 3, 4. 5, 6, 7, 8, 9, or 10;

[0525] each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alkyl; and

[0526] * indicates a bond between a C(=O) carbon and a nitrogen atom of

[0527]

[0528] .

[0529] Embodiment 2 provides the compound of Embodiment 1, wherein each occurrence of L1is independently selected from the group consisting of -(CH2)-, -C(=O)-, and -O-.

[0530] Embodiment 3 provides the compound of Embodiment 1 or 2, wherein -(L1)™- is -C(=O)O(CH2)2-.

[0531] Embodiment 4 provides the compound of any one of Embodiments 1-3, wherein each occurrence of L2is independently selected from the group consisting of -(CH2)2-4-, -N(CH3)-,

[0532] - N---N[(CH2)2-3N(R2d)(R2e)]-, -C(=O)-, -NH-, and

[0533] Embodiment 5 provides the compound of any one of Embodiments 1-4, wherein the compound of formula (I) is selected from the group consisting of:

[0534]

[0535] Attorney Docket No. 046483-7487W01(04056)

[0536]

[0537] Embodiment 6 provides the compound of any one of Embodiments 1-5, wherein R2a. R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of -(CEh)CH(OH)(optionally substituted C1-C22 alkyl).

[0538] Embodiment 7 provides the compound of any one of Embodiments 1-6, wherein R2a, R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from

[0539]

[0540] Embodiment 8 provides the compound of any one of Embodiments 1-7, wherein the small molecule drug comprises at least one primary or secondary amine.

[0541] Embodiment 9 provides the compound of any one of Embodiments 1-8, wherein the small molecule drug is useful for the treatment of cancer.

[0542] Embodiment 10 provides the compound of any one of Embodiments 1-9, wherein the Attorney Docket No. 046483-7487W01(04056)

[0543] small molecule drug is an indoleamine 2,3-dioxygenase (IDO) inhibitor.

[0544] Embodiment 11 provides the compound of Embodiment 10, wherein the IDO inhibitor is indoximod (1-methyl-D-tryptophan).

[0545] Embodiment 12 provides the compound of any one of Embodiments 1-11, wherein

[0546]

[0547] Embodiment 13 provides the compound of any one of Embodiments 1-12, which is selected from the group consisting of 110-SS-l 1-C10, 110-SS-l 1-C12. 110-SS-11-C14, L2A-SS-AA-C10, L2A-SS-AA-C12, L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14, DAB-SS-AA-C10, DAB-SS-AA-C12, DAB-SS-AA-C14, P2A-SS-AA-C10, P2A-SS-AA-C12, P2A-SS-AA-C14, 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14, G0-SS-AA-C10, G0-SS-AA-C12, and G0-SS-AA-C14.

[0548] Embodiment 14 provides a lipid nanoparticle (LNP) composition comprising:

[0549] (a) at least one ionizable lipid;

[0550] (b) at least one neutral lipid;

[0551] (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof, wherein the LNP further comprises:

[0552] (i) at least one nucleic acid encoding an immunomodulatory' protein; and

[0553] (ii) at least one small molecule anticancer agent, wherein the small molecule anticancer agent is attached to at least one component of the LNP by a cleavable linker.

[0554] Embodiment 15 provides the LNP of Embodiment 14, wherein the immunomodulatory protein is interleukin 12 (IL- 12) and the small molecule anticancer agent is indoximod.

[0555] Embodiment 16 provides a lipid nanoparticle (LNP) composition comprising

[0556] (a) at least one ionizable lipid comprising the compound of any one of Embodiments 1-13;

[0557] (b) at least one neutral lipid;

[0558] (c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof, Attorney Docket No. 046483-7487W01(04056)

[0559] wherein the LNP further comprises at least one nucleic acid encoding an immunomodulatory protein.

[0560] Embodiment 17 provides the LNP of Embodiment 16, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 90 mol% of the LNP, optionally wherein the at least one ionizable lipid compound comprises about 50 mol% of the LNP.

[0561] Embodiment 18 provides the LNP of any one of Embodiments 14-17, wherein the at least ionizable lipid compound comprises or consists essentially of at least one ionizable lipid selected from the group consisting of 110-SS-l 1-C10, 110-SS-11 -Cl 2, 110-SS-11-C14, L2A-SS-AA-C10, L2A-SS-AA-C12, L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14, DAB-SS-AA-C10, DAB-SS-AA-C12, DAB-SS-AA-C14, P2A-SS-AA-C10, P2A-SS-AA-C12, P2A-SS-AA-C14. 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14, G0-SS-AA-C10, G0-SS-AA-C12, and G0-SS-AA-C14.

[0562] Embodiment 19 provides the LNP of any one of Embodiments 14-18, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP, optionally wherein the at least one neutral lipid comprises about 10 mol% of the LNP.

[0563] Embodiment 20 provides the LNP of any one of Embodiments 14-19, wherein the neutral lipid comprises or consists essentially of at least one neutral lipid selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC), optionally wherein the neutral lipid comprises or consists essentially of dioleoylphosphatidylethanolamine (DOPE).

[0564] Embodiment 21 provides the LNP of any one of Embodiments 14-20, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 75 mol% of the LNP, optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 38.5 mol% of the LNP.

[0565] Embodiment 22 provides the LNP of any one of Embodiments 14-21, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol.

[0566] Embodiment 23 provides the LNP of any one of Embodiments 14-22, wherein the at least one polymer-conjugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer-conjugated lipid comprises about 1.5 mol%.

[0567] Embodiment 24 provides the LNP of any one of Embodiments 14-23, wherein the at least one polymer-conjugated lipid comprises or consists essentially of 1.2-dimyristoyl-rac-glycero-3-methoxypoly ethylene gly col-2000 (DMG-PEG 2000). Attorney Docket No. 046483-7487W01(04056)

[0568] Embodiment 25 provides the LNP of any one of Embodiments 14-24, wherein the LNP has a molar ratio of (a): (b): (c): (d) of about 50:10:38.5:1.5.

[0569] Embodiment 26 provides the LNP of any one of Embodiments 14-24, wherein the at least one nucleic acid encoding an immunomodulatory protein comprises RNA or DNA.

[0570] Embodiment 27 provides the LNP of any one of Embodiments 14-26, wherein the at least one nucleic acid encoding an immunomodulatory protein comprises mRNA.

[0571] Embodiment 28 provides the LNP of Embodiment 27, wherein the mRNA encodes an interleukin.

[0572] Embodiment 29 provides the LNP of Embodiment 28, wherein the interleukin is interleukin 12 (IL- 12).

[0573] Embodiment 30 provides a pharmaceutical composition comprising the LNP of anyone of Embodiments 14-29 and at least one pharmaceutically acceptable carrier.

[0574] Embodiment 31 provides a method of treating, preventing, and / or ameliorating a disease or disorder in a subject, the method comprising administering to the subject at least one LNP of any one of Embodiments 14-30 or the pharmaceutical composition of Embodiment 30.

[0575] Embodiment 32 provides the method of Embodiment 31, wherein the disease or disorder is at least one selected from the group consisting of cancer, an immune disease or disorder, and a metabolic disease or disorder.

[0576] Embodiment 33 provides the method of Embodiment 31 or 32, wherein the disease or disorder is cancer.

[0577] Embodiment 34 provides the method of Embodiment 33, wherein the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer, CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer.

[0578] The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the Attomey Docket No. 046483-7487W01(04056)

[0579] art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Claims

Attomey Docket No. 046483-7487W01(04056)CLAIMSWhat is claimed is:

1. A compound of formula (I), or a salt, stereoisomer, or isotopologue thereof:wherein:drug;each occurrence of L1is independently selected from the group consisting of -O-, -N(RA)-, and -C(=O)-, and -(optionally substituted C1-C3 alkylenyl)-;each occurrence of L2is independently selected from the group consisting of -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -(optionally substituted C3-C8 cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted C6-C10 arylenyl)-, -(optionally substituted C2-C8 heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-X-, -(optionally substituted C2-C12 alkenylenyl)-X-, -(optionally substituted C1-C12 alkynylenyl)-X-, -(optionally substituted C1-C12 heteroalkylenyl)-X-, -(optionally substituted C3-C8 cycloalkylenyl)-X-, -(optionally substituted C2-C8 heterocyloalkylenyl)-X-, -(optionally substituted Ce-Cio arylenyl)-X-, -(optionally substituted C2-C8 heteroarylenyl)-X-, and -X-;each occurrence of X, if present, is independently selected from the group consisting of -N(R2c)-, -N(R3)-, -N(RA)-, -C(=O)-, and -O-;R1is H;R2a, R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of optionally substituted C1-C24 alkyl and optionally substituted C1-C24 heteroalkyl;each occurrence of R3is independently;each occurrence of L3is independently selected from the group consisting -(optionally substituted C1-C12 alkylenyl)-, -(optionally substituted C2-C12 alkenylenyl)-, -(optionally substituted C1-C12 alkynylenyl)-, -(optionally substituted C1-C12 heteroalkylenyl)-, -Attorney Docket No. 046483-7487W01(04056)(optionally substituted Cs-Cs cycloalkylenyl)-, -(optionally substituted C2-C8 heterocyloalkylenyl)-, -(optionally substituted Ce-Cio arylenyl)-, -(optionally substituted C2- Cs heteroarylenyl)-, -(optionally substituted C1-C12 alkylenyl)-Y-, -(optionally substituted C2- C12 alkenylenyl)-Y-, -(optionally substituted C1-C12 alkynylenyl)-Y-, -(optionally substituted C1-C12 heteroalkylenyl)-Y-, -(optionally substituted C3-C8 cycloalkylenyl)-Y-, -(optionally substituted C2-C8 heterocyloalkylenyl)-Y-, -(optionally substituted Ce-Cio ar lenyl)-Y-. - (optionally substituted C2-C8 heteroarylenyl)-Y-, and -Y-;each occurrence of Y, if present, is independently selected from the group consisting of -N(RA)-, -O-, and -C(=O)-;m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;n is 1, 2, 3, 4. 5, 6, 7. 8, 9, or 10;o is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;each occurrence of RAand RBis independently selected from the group consisting of H and optionally substituted Ci-Ce alky l; and* indicates a bond between a C(=O) carbon and a nitrogen atom of2. The compound of claim 1, wherein each occurrence of L1is independently selected from the group consisting of -(CH2)-, -C(=O)-, and -O-.

3. The compound of claim 1 or 2, wherein -(L1)™- is -C(=O)O(CH2)2-.

4. The compound of any one of claims 1-3, wherein each occurrence of L2is independently selected from the group consisting of -(CH2)2-4-, -N(CH?)-, -N[(CH2)2-5. The compound of any one of claims 1-4, wherein the compound of formula (I) is selected from the group consisting of:Attorney Docket No. 046483-7487W01(04056)6. The compound of any one of claims 1-5. wherein R2'1. R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting of -(CH2)CH(OH)(optionally substituted C1-C22 alkyl).

7. The compound of any one of claims 1-6. wherein R2a. R2b, and each occurrence of R2c, R2d, and R2e, if present, are each independently selected from the group consisting ofAttorney Docket No. 046483-7487W01(04056)8. The compound of any one of claims 1-7. wherein the small molecule drug comprises at least one primary or secondary amine.

9. The compound of any one of claims 1-8, wherein the small molecule drug is useful for the treatment of cancer.

10. The compound of any one of claims 1-9, wherein the small molecule drug is an indoleamine 2,3-dioxygenase (IDO) inhibitor.

11. The compound of claim 10, wherein the IDO inhibitor is indoximod ( 1-methyl-D-tryptophan).

12. The compound of any one of claims 1-11, wherein13. The compound of any one of claims 1-12, which is selected from the group consisting of 110-SS-11-C10, 110-SS-11-C12, 110-SS-11-C14, L2A-SS-AA-C10, L2A-SS-AA-C12, L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14, DAB-SS-AA-C10, DAB-SS-AA-C12, DAB-SS-AA-C14, P2A-SS-AA-C10, P2A-SS-AA-C12, P2A-SS-AA-C14, 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14. G0-SS-AA-C10. GO-SS-AA-C12, and G0-SS-AA-C14.

14. A lipid nanoparticle (LNP) composition comprising:(a) at least one ionizable lipid;(b) at least one neutral lipid;(c) at least one cholesterol lipid and / or a modified derivative thereof; andAttomey Docket No. 046483-7487W01(04056)(d) at least one polymer-conjugated lipid and / or a modified derivative thereof, wherein the LNP further comprises:(i) at least one nucleic acid encoding an immunomodulatory protein; and(ii) at least one small molecule anticancer agent, wherein the small molecule anticancer agent is attached to at least one component of the LNP by a cleavable linker.

15. The LNP of claim 14, wherein the immunomodulatory protein is interleukin 12 (IL-12) and the small molecule anti cancer agent is indoximod.

16. A lipid nanoparticle (LNP) composition comprising:(a) at least one ionizable lipid comprising the compound of any one of claims 1- 13;(b) at least one neutral lipid;(c) at least one cholesterol lipid and / or a modified derivative thereof; and (d) at least one polymer-conjugated lipid and / or a modified derivative thereof, wherein the LNP further comprises at least one nucleic acid encoding an immunomodulatory protein.

17. The LNP of claim 1, wherein the at least one ionizable lipid compound comprises about 10 mol% to about 90 mol% of the LNP, optionally wherein the at least one ionizable lipid compound comprises about 50 mol% of the LNP.

18. The LNP of any one of claims 14-17, wherein the at least ionizable lipid compound comprises or consists essentially of at least one ionizable lipid selected from the group consisting of 110-SS-11-C10, 110-SS-11-C12, 110-SS-11-C14, L2A-SS-AA-C10, L2A-SS-AA-C12, L2A-SS-AA-C14, T3A-SS-AA-C10, T3A-SS-AA-C12, T3A-SS-AA-C14. DAB-SS-AA-C10. DAB-SS-AA-C12, DAB-SS-AA-C14, P2A-SS-AA-C10, P2A-SS-AA-C12, P2A-SS-AA-C14, 306-SS-AA-C10, 306-SS-AA-C12, 306-SS-AA-C14, G0-SS-AA-C10, G0-SS-AA-C12, and G0-SS-AA-C14.

19. The LNP of any one of claims 14-18, wherein the at least one neutral lipid comprises about 1 mol% to about 40 mol% of the LNP, optionally wherein the at least one neutral lipidAttorney Docket No. 046483-7487W01(04056)comprises about 10 mol% of the LNP.

20. The LNP of any one of claims 14-19, wherein the neutral lipid comprises or consists essentially of at least one neutral lipid selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), and dioleoylphosphatidylcholine (DOPC), optionally wherein the neutral lipid comprises or consists essentially of dioleoylphosphatidylethanolamine (DOPE).

21. The LNP of any one of claims 14-20, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 20 mol% to about 75 mol% of the LNP, optionally wherein the at least one cholesterol lipid and / or modified derivative thereof comprises about 38.5 mol% of the LNP.

22. The LNP of any one of claims 14-21, wherein the at least one cholesterol lipid and / or modified derivative thereof comprises or consists essentially of cholesterol.

23. The LNP of any one of claims 14-22, wherein the at least one polymer-conjugated lipid comprises about 0.1 mol% to about 15 mol% of the LNP, optionally wherein the at least one polymer-conjugated lipid comprises about 1.5 mol%.

24. The LNP of any one of claims 14-23, wherein the at least one polymer-conjugated lipid comprises or consists essentially of l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-2000 (DMG-PEG 2000).

25. The LNP of any one of claims 14-24, wherein the LNP has a molar ratio of (a): (b): (c): (d) of about 50:10:38.5:1.5.

26. The LNP of any one of claims 14-24, wherein the at least one nucleic acid encoding an immunomodulatory protein comprises RNA or DNA.

27. The LNP of any one of claims 14-26, wherein the at least one nucleic acid encoding an immunomodulatory protein comprises mRNA.

28. The LNP of claim 27, wherein the mRNA encodes an interleukin.Attomey Docket No. 046483-7487W01(04056)29. The LNP of claim 28, wherein the interleukin is interleukin 12 (IL-12).

30. A pharmaceutical composition comprising the LNP of any one of claims 14-29 and at least one pharmaceutically acceptable carrier.

31. A method of treating, preventing, and / or ameliorating a disease or disorder in a subject, the method comprising administering to the subject at least one LNP of any one of claims 14-30 or the pharmaceutical composition of claim 30.

32. The method of claim 31, wherein the disease or disorder is at least one selected from the group consisting of cancer, an immune disease or disorder, and a metabolic disease or disorder.

33. The method of claim 31 or 32, wherein the disease or disorder is cancer.

34. The method of claim 33, wherein the cancer is at least one selected from the group consisting of pancreatic cancer, colorectal cancer, bladder cancer, breast cancer, prostate cancer, renal cancer, hepatocellular cancer, lung cancer, ovarian cancer, cervical cancer, gastric cancer, esophageal cancer, head and neck cancer, melanoma, neuroendocrine cancer. CNS cancer, brain cancer, bone cancer, soft tissue sarcoma, non-small cell lung cancer, small-cell lung cancer, or colon cancer.

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