Endogenous targeting lipid nanoparticles (ENDO) for systemic delivery of therapeutic agents to pancreas

Abstract

Described herein are methods and lipid nanoparticle compositions for delivering therapeutic agents to the pancreas.

Description

[0001] 208034-0009-W001

[0002] ENDOGENOUS TARGETING LIPID NANOPARTICLES (ENDO) FOR SYSTEMIC DELIVERY OF THERAPEUTIC AGENTS TO PANCREAS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims priority to U.S. Provisional Patent Application No. 63 / 800,917, filed on May 6, 2025, and U.S. Provisional Patent Application No. 63 / 712,809, filed on October 28, 2024, each of which is incorporated by reference herein in its entirety.

[0005] BACKGROUND

[0006] Lipid nanoparticles (LNPs) have revolutionized the field of nucleic acid delivery and have been extensively explored in gene therapy, protein replacement therapies, and immunotherapies for various diseases. Their clinical applications were notably demonstrated by their widespread use in delivering the SARS-CoV-2 vaccine during the COVID-19 pandemic. Despite their promise, targeting beyond the liver remains a significant challenge with the traditional LNPs, which are made up of four key components — ionizable lipid, phospholipid, cholesterol, and polyethylene glycol (PEG) lipid. The accumulation of LNPs in the liver primarily occurs because they associate with Apolipoprotein E (ApoE) in the blood, which is crucial for cholesterol transport and low-density lipoprotein receptor (LDLR)-mediated endocytosis. The mode of administration also plays a critical role in redirecting LNPs to extrahepatic sites. Previously, intraperitoneal administration effectively redirected nanoparticles to the pancreas with robust and specific protein expression via macrophage-mediated gene transfer. Another promising approach for redirecting LNPs to extrahepatic sites is incorporating a fifth component in the traditional LNP formulation. This strategy has been effective in achieving lung- and spleen-specific mRNA expression through interactions with plasma proteins, such as vitronectin (Vtn) and [32 glycoprotein 1 ( 2-GPI), using SORT LNPs containing a fifth component cationic or anionic lipid. Furthermore, the addition of an adjuvant lipidoid containing a Toll-like receptor (TLR) 7 / 8 agonist as a fifth component also successfully demonstrated transfection in the lymph nodes. This illustrates the potential of LNPs to target other hard-to-reach tissues with formulation design, overcoming the inherent limitations of classical four-component LNPs.

[0007] What is needed are endogenous targeting lipid nanoparticles (ENDO) for systemic delivery of therapeutic agents to the pancreas and other tissues. 208034-0009-W001

[0008] SUMMARY

[0009] One embodiment described herein is a lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder. In one aspect, the composition comprises about 10 mol% to about 15 mol% vitamin D or a vitamin D receptor binder. In another aspect, the vitamin D is vitamin D3 (cholecalciferol). In another aspect, the vitamin D receptor binder is a selective antagonist of the vitamin D receptor. In another aspect, the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof. In another aspect, the one or more non-ionizable lipids comprise phospholipids. In another aspect, the phospholipids are selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and combinations thereof. In another aspect, the one or more PEG-modified lipids are selected from the group consisting of 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG 2000), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) (DSPE-PEG), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), and combinations thereof. In another aspect, the composition comprises: about 20 mol% to about 35 mol% ionizable lipids; about 15 mol% to about 17.5 mol% non-ionizable lipids; about 1 mol% to about 4 mol% PEG-modified lipids; and about 40 mol% to about 48 mol% cholesterol or a derivative thereof. In another aspect, the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle. In another aspect, the therapeutic agent comprises one or more nucleic acid molecules. In another aspect, the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof. In another aspect, the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof. In another aspect, the lipid nanoparticle has a diameter size of about 50 nm to about 200 nm. In another aspect, the lipid nanoparticle has a polydispersity index (PDI) of about 0.05 to about 0.3.

[0010] Another embodiment described herein is a method of delivering a lipid nanoparticle composition to a pancreas of a subject, the method comprising: administering to a subject a lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole 208034-0009-W001 percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder. In one aspect, the lipid nanoparticle composition is administered to the subject by intravenous, intraperitoneal, or intramuscular injection. In another aspect, the vitamin D is vitamin D3 (cholecalciferol). In another aspect, the vitamin D receptor binder is a selective antagonist of the vitamin D receptor. In another aspect, the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof. In another aspect, at least 95% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration. In another aspect, at least 99% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration. In another aspect, a portion of the lipid nanoparticle composition is delivered to one or more non-pancreas organs in the subject following administration. In another aspect, the non-pancreas organs comprise one or more muscles, lymph nodes, or a combination thereof in the subject. In another aspect, the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle. In another aspect, the therapeutic agent comprises one or more nucleic acid molecules. In another aspect, the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof. In another aspect, the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof. In another aspect, the method treats, ameliorates, or inhibits the progress of a pancreatic disease in the subject. In another aspect, the pancreatic disease is selected from the group consisting of diabetes, pancreatic cancer, pancreatitis, and combinations thereof. In another aspect, the method results in gene editing, protein replacement, or a combination thereof in the pancreas.

[0011] DESCRIPTION OF THE DRAWINGS

[0012] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0013] FIG. 1A-C show an overview of the fifth component ENDO LN P formulation and screening platform. FIG. 1A shows a schematic representation illustrating the current challenges in developing a robust extrahepatic delivery system. FIG. 1B shows a schematic showing ENDO LNP formulation and screening platform. FIG. 1C shows pie charts depicting the different formulation ratios for ionizable lipid, phospholipid, cholesterol, PEG lipid, and endogenous vitamin fifth component. 208034-0009-W001

[0014] FIG. 2 shows a heatmap representing size (in nm) of ENDO LNPs measured using DLS (n = 3). Abbreviation used: T1, THP1 ; C12, C12-200; SM, SM-102; 306, 306Oi10; F1, Formulation 1 ; F2, Formulation 2; F3, Formulation 3; F4, Formulation 4.

[0015] FIG. 3 shows a heatmap representing PDI of ENDO LNPs measured using DLS (n = 3). Abbreviation used: T1, THP1; C12, C12-200; SM, SM-102; 306, 3060i10; F1 , Formulation 1; F2, Formulation 2; F3, Formulation 3; F4, Formulation 4.

[0016] FIG. 4A-E show in vitro screening of ENDO LNPs. FIG. 4A shows a heat map of in vitro delivery of luciferase mRNA with ENDO LNPs in HEK293 cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after treatment with LNPs. FIG. 4B shows a heat map of in vitro delivery of luciferase mRNA with ENDO LNPs in HFF cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after treatment with LNPs. FIG. 4C shows a heat map of in vitro delivery of luciferase mRNA with ENDO LNPs in HUVEC cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after treatment with LNPs. FIG. 4D shows a heat map of in vitro delivery of luciferase mRNA with ENDO LNPs in RAW 264.7 cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after treatment with LNPs. FIG. 4E shows a heat map of in vitro delivery of luciferase mRNA with ENDO LNPs in HMC3 cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after treatment with LNPs. Abbreviation used: T1 , THP1 ; C12, C12-200; SM, SM-102; 306, 3060i10; F 1 , Formulationl ; F 2, Formulation2; F 3, Formulations; F 4, Formulation4.

[0017] FIG. 5A-F shows characterization and transfection efficiency of traditional four component control LNPs. FIG. 5A shows the size (nm), polydispersity index (PDI), and encapsulation efficiency (EE%) of traditional four component control LNPs. FIG. 5B shows in vitro delivery efficacy of FLuc mRNA traditional four component LNPs in HEK293 cells (125 ng mRNA per well, 96 well plate, n = 3). FIG. 5C shows in vitro delivery efficacy of FLuc mRNA traditional four component LNPs in HFF cells (125 ng mRNA per well, 96 well plate, n = 3). FIG. 5D shows in vitro delivery efficacy of FLuc mRNA traditional four component LNPs in HMC3 cells (125 ng mRNA per well, 96 well plate, n = 3). FIG. 5E shows in vitro delivery efficacy of FLuc mRNA traditional four component LNPs in HUVEC cells (125 ng mRNA per well, 96 well plate, n = 3). FIG. 5F shows in vitro delivery efficacy of FLuc mRNA traditional four component LNPs in RAW264.7 cells (125 ng mRNA per well, 96 well plate, n = 3). Luminescence intensity was quantified 24 h after adding LNPs.

[0018] FIG. 6A-C show in Vivo screening of ENDO LNPs using batch analysis. FIG. 6A shows IVIS images at 24 h post-injection and graphical representation of total flux of FLuc mRNA ENDO 208034-0009-W001

[0019] LNP pool based on formulations (F1 , F2, F3 and F4). SM-102 and MC3 were used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg / kg of pooled ENDO LNPs. (n = 2 biologically independent mice, ± SD). FIG. 6B shows I VIS images at 24 h post-injection and graphical representation of total flux of FLuc mRNA ENDO LNP pool based on ionizable lipids. Pancreas targeting F2 and F3 LNPs were pooled and batched based on the respective ionizable lipids (THP1 , C12-200, SM-102, MC3, 3060110) and injected intravenously. SM-102 and MC3 were used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg / kg of pooled ENDO LNPs. (n = 2 biologically independent mice, ± SD). FIG. 6C shows I VIS images at 24 h post-injection and graphical representation of total flux of FLuc mRNA ENDO LNP pool based on endogenous vitamins. Selective pancreas targeting C 12-200 ENDO LNPs were then batched based on vitamins A (retinol), B2 (riboflavin), D3 (cholecalciferol), E (tocopherol), and K1 (phylloquinone). SM-102 and MC3 were used as a control. C57BL / 6 mice were injected intravenously with 0.5 mg / kg of pooled ENDO LNPs. (n = 2 biologically independent mice, ± SD).

[0020] FIG. 7A-D show validation of top ENDO LNPs, C-CholF2 and C-CholF3. FIG. 7A shows physiochemical properties such as size, PDI, ^-potential measurements, and mRNA encapsulation efficiency of C-CholF2 and C-CholF3 along with control (C12-200). (n = 3, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****p < 0.0001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). FIG. 7B shows representative I VI S images at 24 h post-injection and graphical representation of total flux of C-CholF2 and C-CholF3 ENDO LNPs injected intravenously at a dose of 0.5 mg / kg. PBS and C12-200 were also injected as a control (n = 3 biologically independent mice, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****p < 0.0001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). FIG. 7C shows pie charts illustrating the percentage of protein expression occurring per organ after intravenous injection of C12-200 (four component traditional components) and redirection to the pancreas for C-CholF2 and C-CholF3 LNPs (n = 3 biologically independent mice). FIG. 7B shows representative cryo- EM image of C-CholF3 LNPs. Scale bar, 50 nm.

[0021] FIG. 8 shows replicates of I VIS images from FIG. 7B after administration of C-CholF2 and C-CholF3 LNPs injected intravenously at a dose of 0.5 mg / kg. PBS and C12-200 were also injected (n = 3 biologically independent mice).

[0022] FIG. 9 shows representative cryo-EM image of C-CholF3 LNPs. Scale bar, 50 nm.

[0023] FIG. 10 shows graphical representation of total flux at 24h post-injection of different doses (0.25, 0.5, and 1 mg / kg) of C-CholF3 LNPs administered intravenously in C57BL / 6 mice (n = 3 biologically independent mice). 208034-0009-W001

[0024] FIG. 11A-C show toxicity and safety evaluation of C-CholF3 ENDO LNPs. FIG. 11A shows representative histology images of the pancreas (24 h and 48 h post- treatment) and liver sections 48 h post-treatment with C-CholF3 encapsulating FLuc mRNA, and PBS (control) via intravenous injection in C57BL / 6 mice at a dose of 0.5 mg / kg. Hematoxylin and Eosin (H&E) staining was performed, with images taken at 40* magnification. Scale bars: 60 m (n = 3 biologically independent mice). FIG. 11 B shows serum levels of liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST), renal parameters, blood urea nitrogen (BUN), and creatinine 24 h after intravenous administration with PBS, C-CholF3 and C12-200 LNPs at a dose of 0.5 mg / kg (n = 3, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001 ; NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). FIG. 11C shows levels of IL-6, IL-1 [3, and TNFa in mice intravenously treated with C-CholF3 and C12-200 LNPs at a dose of 0.5 mg / kg (n = 3 biologically independent mice, ± SD, *P< 0.05, **P< 0.01 , ***p< 0.001 ; NS indicates no significance, one-way ANOVA with Bonferroni post-hoc analysis). PBS- injected mice were kept as the control group.

[0025] FIG. 12 shows replicates of H&E staining of the pancreas after 24 h used to generate FIG. 11A.

[0026] FIG. 13 shows replicates of H&E staining of the pancreas and liver after 24 h used to generate FIG. 11 A.

[0027] FIG. 14 shows the body weight of mice treated with C-CholF3 and C12-200 LNPs at a dose of 0.5 mg / kg intravenously for 21 days (n = 3). PBS was intravenously injected into mice as a control.

[0028] FIG. 15 shows serum levels of the liver enzyme alkaline phosphatase 24 h after intravenous administration with PBS, C-CholF3, and C12-200 LNPs at a dose of 0.5 mg / kg. (n = 3 biologically independent mice, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001 , ****P < 0.0001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis).

[0029] FIG. 16 shows levels of biomarkers GM-CSF, IFNy, IL-2, IL-4, IL-10, IL-12P70, and MCP- 1 (24 h post-treatment) in mice intravenously treated with C-CholF3 and C12-200 LNPs at a dose of 0.5 mg / kg (n = 3 biologically independent mice, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). PBS-injected mice were kept as the control group.

[0030] FIG. 17A-B shows stability study of C-CholF3 LNPs. FIG. 17A shows size, PDI, and - potential of C-CholF3 LNPs under different storage conditions (4 °C and -20 °C) for 1 , 3, 7, and 21 days. FIG. 17B shows representative IVIS images of total flux 24 h after intravenous injection 208034-0009-W001 of FLuc mRNA-loaded C-CholF3 LNPs after being stored at different temperatures for extended time (0.5 mg / kg, n = 3).

[0031] FIG. 18 shows IVIS images and graphical representation of in vivo kinetics of FLuc expression following intravenous injection of C-CholF3 LNPs at a dose of 0.5 mg / kg (n = 3 biologically independent mice, ± SD). The luciferase expression was visualized at 24, 48, and 72 hours after injection by IVIS.

[0032] FIG. 19 shows TNS curves for pKa measurements of C-CholF3 and C12-200 LNPs.

[0033] FIG. 20A-B show C-CholF3 can deliver plasmid DNA (pDNA) and circular mRNA (mRNA) to the pancreas in vivo. FIG. 20A shows representative IVIS images at 24 h post-injection and graphical representation of total flux of C-CholF3 ENDO LNPs formulated with pDNA and circular mRNA injected intravenously at a dose of 0.5 mg / kg (n = 3 biologically independent mice). FIG. 20B shows pie charts illustrating the percentage of protein expression occurring per organ after intravenous injection of C-CholF3 ENDO LNPs formulated with pDNA and circular mRNA (n = 3 biologically independent mice).

[0034] FIG. 21A-F show mechanistic insights into endogenous targeting of C-CholF3 ENDO LNPs to the pancreas. FIG. 21 A shows confocal microscopy images showing the cellular uptake and intracellular trafficking of DiO-labeled C-CholF3 and MC3 LNPs in BxPC-3 cells. Cells were stained with LysoTracker Red (endosomes / lysosomes) and Hoechst 33342 (nuclei) and imaged

[0035] 2 h post-treatment to visualize nanoparticle localization. Scale bars are 10 pm. Images were captured at 60* magnification. FIG. 21 B shows schematic illustration of experimental design of in vitro Vitamin D Receptor (VDR) corona study. C-CholF3 mRNA LNPs were preincubated with recombinant VDR protein prior to treatment in BxPC-3 cells to assess enhanced mRNA delivery. FIG. 21C shows in vitro transfection efficiency of C-CholF3 LNPs pre-coated with VDR in BxPC-

[0036] 3 cells. Cells were treated with 125 ng luciferase mRNA per well in a 96-well plate (n = 5), and luminescence intensity was quantified 24 h post-treatment. FIG. 21 D shows schematic representation of VDR blockade using MeTC7. Mice were administered 50 mg / kg of MeTC7 intraperitoneally, and 12 hours later, mRNA LNPs were administered intravenously. To investigate the role of VDR, mice were pre-administering with VDR antagonist MeTC7 or PBS (control) 12 h before LNP administration. FIG. 21 E shows representative IVIS images at 24 h post-injection of LNPs (0.5 mg / kg) in C57BL / 6 mice (n = 4 biologically independent mice). FIG. 21 F shows representative IVIS images at 24 h post-injection of C-CholF3 LNPs formulated with MeTC7 as the fifth component in place of cholecalciferol. Mice were treated intravenously at a dose of 0.5 mg / kg (n = 3 biologically independent mice). 208034-0009-W001

[0037] FIG. 22 shows replicates of confocal microscope images to generate FIG. 21 A. Confocal images of BxPC-3 treated with DiO tagged C-CholF3 ENDO LNPs (green). Cells were stained with LysoTracker Red (endosomes / lysosomes) and Hoechst 33342 (nuclei). Scale bars are 10 pm. Images were captured at 63* magnification.

[0038] FIG. 23 shows replicates of confocal microscope images to generate FIG. 21 A. Confocal images of BxPC-3 treated with DiO tagged MC3 LNPs (green). Cells were stained with LysoTracker Red (endosomes / lysosomes) and Hoechst 33342 (nuclei). Scale bars are 10 pm. Images were captured at 63x magnification.

[0039] FIG. 24 shows replicates of confocal microscope images to generate FIG. 21 A. Confocal images of untreated BxPC-3 cells. Cells were stained with LysoTracker Red (endosomes / lysosomes) and Hoechst 33342 (nuclei). Scale bars are 10 pm. Images were captured at 63x magnification.

[0040] FIG. 25 shows replicates of IVIS images from FIG. 21 D showing luciferase expression 24 h after intravenous administration of C-CholF3 and MC3 mRNA LNPs (0.5 mg / kg) in C57BL / 6 mice. Mice were pretreated intraperitoneally with either 50 mg / kg MeTC7 (VDR antagonist) or PBS (control) 12 h before LNP administration (n = 4 biologically independent mice).

[0041] FIG. 26A-B show MeTC7 as a fifth component in pancreas targeting formulation can retain pancreas tropism in vivo. FIG. 26A shows replicates of IVIS images from FIG. 6E and graphical representation of total flux of C-CholF3 LNPs and LNPs with MeTC7 as the fifth component 24 h post-injection. Mice were treated intravenously at a dose of 0.5 mg / kg (n = 3 biologically independent mice). FIG. 26B shows pie charts illustrating the percentage of protein expression occurring per organ after intravenous injection of C-CholF3 LNPs and LNPs with MeTC7 as the fifth component (n = 3 biologically independent mice).

[0042] FIG. 27A-D show efficient and tissue-specific tdTomato expression in the pancreas with C-CholF3 ENDO LNPs. FIG. 27A shows a schematic illustration of Cre mRNA delivery and subsequent Cre-mediated removal of the stop cassette, leading to the activation of tdTomato expression in the Ai 14 Cre-loxP mouse model. LNPs formulated with Cre mRNA were injected intravenously and the pancreas was imaged using the IVIS. FIG. 27A shows representative tdTomato expression in the pancreas 120 h post-injection and graphical representation of total flux of C-CholF3 LNPs containing Cre mRNA, and PBS treated control injected intravenously to Ai14 mice at a dose of 1.5 mg / kg (n = 4 biologically independent mice, ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis). FIG. 27C shows pie charts illustrating the percentage of protein expression occurring in pancreas and liver after intravenous injection of Cre mRNA containing C-CholF3 LNPs. (n = 4 biologically 208034-0009-W001 independent mice). FIG. 27D shows representative immunofluorescent images of pancreatic sections 120 h post-injection with C-CholF3 LNPs encapsulating Cre mRNA and with PBS (control) via intravenous injection in Ai14 mice at a dose of 1.5 mg / kg. Insulin antibody was used to stain the -cells, and DAPI was used to label the nuclei. Images were captured at 40x magnification from three biologically independent mice.

[0043] FIG. 28 shows representative tdTomato expression in pancreas and liver 120h postinjection of C-CholF3 LNPs containing Cre mRNA, and PBS treated control injected intravenously to Ai14 mice at a dose of 1.5 mg / kg (n = 4 biologically independent mice, ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post-hoc analysis).

[0044] FIG. 29 shows replicates of images used to generate FIG. 27D. Immunofluorescent images of pancreatic sections from PBS treatment Ai14 mice. Insulin antibody was used to stain the p-cells, and DAPI was used to label the nuclei. Images were captured at 40* magnification from three biologically independent mice.

[0045] FIG. 30 shows replicates of images used to generate FIG. 27D. Immunofluorescent images of pancreatic sections from C-CholF3 LNPs treated Ai14 mice at a dose of 1.5 mg / kg. Insulin antibody was used to stain the -cells, and DAPI was used to label the nuclei. Images were captured at 40* magnification from three biologically independent mice.

[0046] FIG. 31A-B show comparisons of protein expression of C-CholF3 ENDO LNPs with MC3 LNPs. FIG. 31 A shows representative I VIS images at 4h post injection and graphical representation of total flux of C-CholF3 and MC3 mRNA LNPs injected intravenously at a dose of 0.5 mg / kg (n= 3 biologically independent mice). FIG. 31 B shows representative MS images at 24h post injection and graphical representation of total flux of C-CholF3 and MC3 mRNA LNPs injected intravenously at a dose of 0.5 mg / kg (n= 3 biologically independent mice).

[0047] FIG. 32A-C show comparisons of toxicity and safety evaluation of C-CholF3 ENDO LNPs with MC3 LNPs. FIG. 32A shows serum levels of liver enzymes, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and Alkaline Phosphatase after intravenous administration with C-CholF3 and MC3 mRNA LNPs at 4 h and 24h (0.5 mg / kg, n = 3, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post hoc analysis). FIG. 32B shows renal parameters, blood urea nitrogen (BUN), and creatinine after intravenous administration with C-CholF3 and MC3 mRNA LNPs at 4 h and 24h (0.5 mg / kg, n = 3, ± SD, *P < 0.05, **P < 0.01, ***P < 0.001. NS, not significant, one-way ANOVA with Bonferroni post hoc analysis). FIG. 32C shows levels of IL-6, IL-1 p, TNFa, GM-CSF, IFNg, IL-2, IL-4, IL-10, I L-12p70 and MCP-1 in mice after intravenous administration with C-CholF3 and MC3 mRNA LNPs at 4 h 208034-0009-W001 and 24h (0.5 mg / kg, n = 3, ± SD, *P < 0.05, **P < 0.01 , ***P < 0.001. NS, not significant, oneway A NOVA with Bonferroni post hoc analysis).

[0048] FIG. 33A-C show in vivo intramuscular screening of ENDO LNPs. FIG. 33A shows IVIS images at 6 h post injection and graphical representation of total flux of FLuc mRNA encapsulated with all ENDO LNPs, batched based on formulation ratios. Traditional four component DLin-MC3- DMA (MC3) and SM-102 mRNA LNPs were used as controls. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled ENDO nanoparticles (n = 2 biologically independent mice, initial screening). FIG. 33B shows IVIS images at 6 h post injection and graphical representation of total flux of FLuc mRNA encapsulated with all ENDO LNPs, batched based on ionizable lipids from formulation 1 (F1) and 2 (F2). Traditional four component DLin-MC3-DMA (MC3) and SM-102 mRNA LNPs were used as controls. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled ENDO nanoparticles (n = 2 biologically independent mice, initial screening). FIG. 33C shows IVIS images at 6 h post injection and graphical representation of total flux of FLuc mRNA encapsulated with all ENDO LNPs, batched based on vitamins from ionizable lipid C12-200. Traditional four component DLin-MC3-DMA (MC3) and SM-102 mRNA LNPs were used as controls. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled ENDO nanoparticles (n = 2 biologically independent mice, initial screening).

[0049] FIG. 34A-B show validation of top hits from the batch analysis. FIG. 34A shows IVIS images at 6 h post injection and graphical representation of total flux of FLuc mRNA encapsulated in C-RetF1 , C-RetF2, C-TocF1 and C-TocF2 ENDO LNPs. Traditional four component C12-200 and SM-102 mRNA LNPs were used as controls. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled ENDO nanoparticles (n = 3 biologically independent mice, initial screening). FIG. 34B shows ex vivo IVIS images of organs (liver, spleen, and lymph nodes) at 6 h post injection and graphical representation of total flux of FLuc mRNA encapsulated in C-RetF1 , C-RetF2, C-TocF1 and C-TocF2 ENDO LNPs. Traditional four component C12-200 and SM-102 mRNA LNPs were used as controls. C57BL / 6 mice were injected intramuscularly with 0.5 mg / kg of pooled ENDO nanoparticles (n = 3 biologically independent mice, initial screening).

[0050] DETAILED DESCRIPTION

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the 208034-0009-W001 present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0052] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0053] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of” the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0054] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0055] As used herein, the term “or” can be conjunctive or disjunctive.

[0056] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0057] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0058] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.” 208034-0009-W001

[0059] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0060] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0061] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0062] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0063] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0064] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0065] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a 208034-0009-W001 disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0066] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0067] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments.

[0068] As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0069] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of’ or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0070] Lipid nanoparticles (LNPs) hold transformative potential for nucleic acid delivery, with applications ranging from clinical use, particularly in COVID-19 vaccines, to gene therapy and cancer immunotherapy. However, a major limitation lies in their preferential accumulation in the liver following intravenous administration, making most targets hard-to-reach. Described herein is a novel platform called endogenous targeting lipid nanoparticles (ENDO), containing, in some aspects, cholecalciferol (vitamin D3) as a fifth component, that selectively delivers therapeutic agents (e.g., mRNA) to the pancreas - a target previously inaccessible through intravenous 208034-0009-W001 administration. One promising formulation, C-CholF3, demonstrates an unprecedented 99% pancreas selectivity with robust and sustained protein expression for up to 3 days in a dosedependent manner with minimal toxicity that makes it suitable for repeat administration. We propose that this organ-specific delivery is mediated through an endogenous targeting mechanism involving the Vitamin D receptor (VDR). C-CholF3 also enables selective pancreatic delivery of plasmid DNA and circular mRNA, underscoring its versatility and therapeutic potential. Furthermore, C-CholF3 exhibits pancreas-specific gene editing in the Ai14 transgenic mouse model, showing high expression of tdTomato in the > cells. These findings highlight its potential for translational applications in protein replacement and CRISPR / Cas9-mediated gene editing for currently incurable pancreatic diseases, including pancreatic cancer and diabetes.

[0071] Described herein is a novel platform called endogenous targeting lipid nanoparticles (ENDO), which incorporates endogenous ligands such as vitamins as the fifth component in the LNP formulation, fine-tuning their organ tropism and enhancing targeted delivery (FIG. 1A). We chose endogenous vitamins because of their distinct chemical structures and unique biochemical functions, which make them ideal candidates for the fifth component platform. Structurally, vitamins can be broadly divided into water-soluble groups, such as the vitamin B complex, and fat-soluble groups, which include vitamins A, D, E, and K. The fat-soluble vitamins are particularly relevant to the LNP formulations due to their hydrophobic tails, which can integrate seamlessly into the lipid bilayer. Additionally, ionizable lipids containing vitamin C have been shown to improve transfection efficiency and exhibit anti-microbial properties against sepsis. In this study, we screened a library of 100 LNPs formulated with various combinations of fifth-component endogenous vitamins, different ionizable lipids, and distinct formulation ratios to assess their potential for tissue-specific delivery (FIG. 1 B). The resulting ENDO LNPs were tested in five different cell lines representing major cell types in the body and demonstrated varying levels of transfection across these cell types. We further investigated the ability of these LNPs to deliver mRNA in vivo using batch analysis.

[0072] This screening led to the identification of two formulations containing cholecalciferol as a fifth component, which demonstrated selective delivery of mRNA to the pancreas with high efficacy through intravenous administration - C-CholF2 and C-CholF3. Among these formulations, C-CholF3 exhibited higher protein expression than C-CholF2, achieving over 99% selectivity and sustained expression in the pancreas for up to 3 days in a dose-dependent manner. We propose that the pancreas tropism of C-CholF3 arises from an endogenous targeting mechanism involving the Vitamin D receptor (VDR), based on our corona study and loss of pancreatic protein expression following VDR blockade using MeTC7 antagonist. In addition to 208034-0009-W001 mRNA, C-CholF3 facilitates selective pancreatic delivery of other nucleic acid cargos, including both plasmid DNA and circular mRNA, establishing broadly compatible nucleic acid delivery platform for diverse therapeutic applications. To our knowledge, no existing material or LNP formulation has demonstrated the capability to deliver mRNA specifically to the pancreas via intravenous administration. The addition of biocompatible vitamins also resulted in reduced toxicity, improved safety profiles, and enhanced tolerance. Furthermore, we assessed the gene editing ability of C-CholF3 LNP in the Ai14 mouse model and observed tissue-specific gene editing in the pancreas, with efficient expression of tdTomato in cells. In summary, the ENDO platform highlights the importance of incorporating endogenous vitamins as the fifth component of LNPs for targeted delivery to specific tissues. Furthermore, it broadens the potential for targeting hard-to-reach extrahepatic organs, such as the pancreas, and provides solutions for protein replacement and CRISPR / Cas9-mediated gene editing to treat currently incurable pancreatic diseases.

[0073] One embodiment described herein is a lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder. In one aspect, the composition comprises about 10 mol% to about 15 mol% vitamin D or a vitamin D receptor binder. In another aspect, the vitamin D is vitamin D3 (cholecalciferol). In another aspect, the vitamin D receptor binder is a selective antagonist of the vitamin D receptor. In another aspect, the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof. In another aspect, the one or more non-ionizable lipids comprise phospholipids. In another aspect, the phospholipids are selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and combinations thereof. In another aspect, the one or more PEG-modified lipids are selected from the group consisting of 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG- PEG 2000), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol) (DSPE-PEG), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG-DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), and combinations thereof. In another aspect, the composition comprises: about 20 mol% to about 35 mol% ionizable lipids; about 15 mol% to about 17.5 mol% non-ionizable lipids; about 1 mol% to about 4 mol% PEG-modified 208034-0009-W001 lipids; and about 40 mol% to about 48 mol% cholesterol or a derivative thereof. In another aspect, the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle. In another aspect, the therapeutic agent comprises one or more nucleic acid molecules. In another aspect, the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof. In another aspect, the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof. In another aspect, the lipid nanoparticle has a diameter size of about 50 nm to about 200 nm. In another aspect, the lipid nanoparticle has a polydispersity index (PDI) of about 0.05 to about 0.3.

[0074] Another embodiment described herein is a method of delivering a lipid nanoparticle composition to a pancreas of a subject, the method comprising: administering to a subject a lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder. In one aspect, the lipid nanoparticle composition is administered to the subject by intravenous, intraperitoneal, or intramuscular injection. In another aspect, the vitamin D is vitamin D3 (cholecalciferol). In another aspect, the vitamin D receptor binder is a selective antagonist of the vitamin D receptor. In another aspect, the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof. In another aspect, at least 95% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration. In another aspect, at least 99% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration. In another aspect, a portion of the lipid nanoparticle composition is delivered to one or more non-pancreas organs in the subject following administration. In another aspect, the non-pancreas organs comprise one or more muscles, lymph nodes, or a combination thereof in the subject. In another aspect, the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle. In another aspect, the therapeutic agent comprises one or more nucleic acid molecules. In another aspect, the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof. In another aspect, the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof. In another aspect, the method treats, ameliorates, or inhibits the progress of a pancreatic disease in the subject. In another aspect, the pancreatic disease is selected from the group consisting of diabetes, pancreatic cancer, 208034-0009-W001 pancreatitis, and combinations thereof. In another aspect, the method results in gene editing, protein replacement, or a combination thereof in the pancreas.

[0075] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0076] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0077] Clause 1. A lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder.

[0078] Clause 2. The composition of clause 1 , wherein the composition comprises about 10 mol% to about 15 mol% vitamin D or a vitamin D receptor binder. 208034-0009-W001

[0079] Clause 3. The composition of clause 1 or 2, wherein the vitamin D is vitamin D3 (cholecalciferol).

[0080] Clause 4. The composition of any one of clauses 1-3, wherein the vitamin D receptor binder is a selective antagonist of the vitamin D receptor.

[0081] Clause 5. The composition of any one of clauses 1-4, wherein the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof.

[0082] Clause 6. The composition of any one of clauses 1-5, wherein the one or more non-ionizable lipids comprise phospholipids.

[0083] Clause 7. The composition any one of clauses 1-6, wherein the phospholipids are selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and combinations thereof.

[0084] Clause 8. The composition of any one of clauses 1-7, wherein the one or more PEG- modified lipids are selected from the group consisting of 1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol-2000 (DMG-PEG 2000), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol) (DSPE-PEG), 1 ,2-distearoyl-sn- glycero-3-phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG- DSPE), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (14:0 PEG2000 PE), and combinations thereof.

[0085] Clause 9. The composition of any one of clauses 1-8, wherein the composition comprises: about 20 mol% to about 35 mol% ionizable lipids; about 15 mol% to about 17.5 mol% non-ionizable lipids; about 1 mol% to about 4 mol% PEG-modified lipids; and about 40 mol% to about 48 mol% cholesterol or a derivative thereof.

[0086] Clause 10. The composition of any one of clauses 1-9, further comprising a therapeutic agent encapsulated in the lipid nanoparticle.

[0087] Clause H . The composition any one of clauses 1-10, wherein the therapeutic agent comprises one or more nucleic acid molecules.

[0088] Clause 12. The composition of any one of clauses 1-11 , wherein the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof.

[0089] Clause 13. The composition of any one of clauses 1-12, wherein the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof. 208034-0009-W001

[0090] Clause 14. The composition of any one of clauses 1-13, wherein the lipid nanoparticle has a diameter size of about 50 nm to about 200 nm.

[0091] Clause 15. The composition any one of clauses 1-14, wherein the lipid nanoparticle has a polydispersity index (PDI) of about 0.05 to about 0.3.

[0092] Clause 16. A method of delivering a lipid nanoparticle composition to a pancreas of a subject, the method comprising: administering to a subject a lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder.

[0093] Clause 17. The method of clause 16, wherein the lipid nanoparticle composition is administered to the subject by intravenous, intraperitoneal, or intramuscular injection.

[0094] Clause 18. The method of clause 16 or 17, wherein the vitamin D is vitamin D3

[0095] (cholecalciferol).

[0096] Clause 19. The method of any one of clauses 16-18, wherein the vitamin D receptor binder is a selective antagonist of the vitamin D receptor.

[0097] Clause 20. The method of any one of clauses 16-19, wherein the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof.

[0098] Clause 21. The method of any one of clauses 16-20, wherein at least 95% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration.

[0099] Clause 22. The method of any one of clauses 16-21 , wherein at least 99% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration.

[0100] Clause 23. The method of any one of clauses 16-22, wherein a portion of the lipid nanoparticle composition is delivered to one or more non-pancreas organs in the subject following administration. 208034-0009-W001

[0101] Clause 24. The method of any one of clauses 16-23, wherein the non-pancreas organs comprise one or more muscles, lymph nodes, or a combination thereof in the subject.

[0102] Clause 25. The method of any one of clauses 16-24, wherein the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle.

[0103] Clause 26. The method of any one of clauses 16-25, wherein the therapeutic agent comprises one or more nucleic acid molecules.

[0104] Clause 27. The method of any one of clauses 16-26, wherein the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof.

[0105] Clause 28. The method of any one of clauses 16-27, wherein the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof.

[0106] Clause 29. The method of any one of clauses 16-28, wherein the method treats, ameliorates, or inhibits the progress of a pancreatic disease in the subject.

[0107] Clause 30. The method of any one of clauses 16-29, wherein the pancreatic disease is selected from the group consisting of diabetes, pancreatic cancer, pancreatitis, and combinations thereof.

[0108] Clause 31. The method of any one of clauses 16-30, wherein the method results in gene editing, protein replacement, or a combination thereof in the pancreas.

[0109] EXAMPLES

[0110] Example 1

[0111] Endogenous Targeting Lipid Nanoparticles (ENDO) for Systemic Delivery of mRNA to Pancreas Materials

[0112] 1 ,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol and 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K) were purchased from Avanti Polar Lipids, DLin-MC3-DMA, SM-102, 3060i10, C12-200, Vitamin A, Vitamin D3, Vitamin K1 , and (±)-a-Tocopherol was purchased from Cayman Chemicals. Riboflavin and Triton X-100 was purchased from Sigma. THP1 was synthesized in our lab using our established method. See Isaac et al., ACS Nano 18: 29045 (2024). Fetal Bovine serum was purchased from Gibco. QuantiT RiboGreen RNA Assay Kit was purchased from Invitrogen. Clean cap FLuc- mRNA and CleanCap Cre mRNA was purchased from Trilink Biotechnology. HEK 293, BxPC-3 HUVEC and RAW 264.7 cell lines purchased from ATCC. HFF and HMC3 cell lines were given by Dr. Seungman Park’s lab at UNLV All the cells were cultured according to the ATCC guidelines. DMEM, RPMI and MEM growth medium (Gibco, USA) containing sodium 208034-0009-W001 bicarbonate, without sodium pyruvate and HEPES, was supplemented with 10% fetal bovine serum (Gibco, USA) and 1 % penicillin / streptomycin (Thermo Fisher Scientific). Human umbilical vein endothelial cells were maintained in Ham’s F12K medium (ATCC, USA) supplemented with 10% fetal bovine serum, 1 % penicillin-streptomycin-amphotericin B (Fungizone) mix (BioWhittaker, Walkersville, Md.), 30 pg of endothelial cell growth supplement per ml, and 100 pg of heparin (Sigma) per ml. MeTC7 was purchased from MedChemExpress (New Jersey, USA). Recombinant Human Vitamin D Receptor was purchased from NovateinBio (Massachusetts, USA).

[0113] Formulation of Lipid Nanoparticles

[0114] LNPs were formulated using previously established protocol. See Kauffman et al., Nano Lett. 15: 7300 (2015). Briefly, the organic phase was prepared by dissolving DOPE and DMG- PEG200 in ethanol at a molar ratio of 16 and 2.5 respectively. The molar ratios of ionizable lipids, cholesterol and vitamins vary by formulation and range from 20 to 35% for ionizable lipids, 41.5 to 46.5% for Cholesterol and 5 to 15% for the fifth component. The generic four-component LNPs (SM-102, MC3, C12-200) which were used as controls were formulated in a molar ratio of 35:16:46.5:2.5. The aqueous phase was prepared by dissolving the corresponding mRNA in 10 mM citrate buffer at pH 3 (Teknova, Hollister, CA, USA). The ionizable lipid to mRNA weight ratio for all LNPs was 10:1.50. The hundred formulations were formulated in a 96-well plate, where each well had the ethanol phase and the aqueous phase was mixed rapidly in the well using a multichannel pipette. During validations, the two phases were loaded into separate glass syringes (Hamilton Company, Reno, NV) and LNPs were formed by chaotic mixing of the organic and aqueous phases at a 1:3 volume ratio in a microfluidic device using Fusion 400 X (Chemyx Inc, USA). The LNPs were subsequently dialyzed against 1 x PBS (Thermo Fisher Scientific, Walthman, MA, USA) in 20 kDa molecular weight cutoff dialysis cassettes (Thermo Fisher Scientific) for 4 hours.

[0115] Characterization of Lipid Nanoparticles

[0116] The encapsulated mRNA concentration and encapsulation efficiency of the LNPs were assessed using the Quant-iT RiboGreen assay (Thermo Fisher Scientific), following established protocols. See e.g., Reinhart et al., Mol. Pharmaceutics 20: 6492 (2023). Each LNP sample was diluted 100-fold in two microcentrifuge tubes, one containing 1 x TE buffer and the other containing 1% (v / v) Triton X-100 (Sigma, USA) in 1x TE buffer. The Triton X-100 samples were mixed thoroughly and incubated for 5 minutes to lyse the LNPs. Samples (LNPs in 1 x TE buffer, LNPs 208034-0009-W001 in 1% Triton X-100, and mRNA standards) were placed in quadruplicate in black-walled 96-well plates. The RiboGreen detection reagent was then added to each well according to the manufacturer’s instructions. The plate was shaken at 200 rpm in the dark for 5 minutes, and fluorescence intensity was measured using a GloMax Explorer plate reader (Promega, USA) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The encapsulated mRNA concentration was calculated from a standard curve generated using univariate leastsquares linear regression. Encapsulation efficiency (EE) was determined using the formula: where RTE is the free RNA content in TE buffer, and RTX is the total RNA content in 1% Triton X- 100 buffer. The hydrodynamic diameter and polydispersity index (PDI) of the LNPs were measured using a Mobius instrument (Wyatt Technology, Santa Barbara, CA, USA). Each LNP sample was diluted 100-fold in 1 x PBS and placed in a cuvette (Wyatt Technology) for analysis. The surface ^-potential was also measured using the Mobius, with each LNP sample diluted 100- fold in deionized water (Thermo Fisher Scientific) and placed into a capillary cell for measurement. Size and ^-potential were reported as mean ± standard deviation (n = 3 technical replicates).

[0117] TNS Assay

[0118] The pKa of LNPs was determined using a TNS (6-(p-Toluidino)-2-naphthalenesulfonic acid) binding assay. A stock solution of 0.16 mM TNS reagent (Sigma Aldrich) was prepared in deionized water. LNPs were diluted to a concentration of 40 ng / mL, and 10 pL of the TNS stock solution was added to each well. The final volume per well was adjusted to 250 pL, consisting of 150 mM sodium chloride, 20 mM sodium phosphate, 20 mM ammonium acetate, and 25 mM ammonium citrate. The assay was conducted across a pH range of 2 to 12, with increments of 0.5 pH units. Samples were placed in black 96-well plates and mixed on a plate shaker at 300 rpm for 5 minutes at room temperature in the dark. Fluorescence measurements were taken using a GloMax Explorer plate reader (Promega), with excitation and emission wavelengths set at 322 nm and 431 nm, respectively, each with a 20 nm bandwidth and a gain of 60. Normalized fluorescence data were plotted against pH, and the pKa value was determined as the pH corresponding to the inflection point of the titration curve.

[0119] In Vitro Studies

[0120] Cells were seeded at a density of 18,000 cells per well in 100 pL of DMEM in a 96-well plate and allowed to adhere for 24 hours. After this, the media was removed, and 75 pL of fresh 208034-0009-W001

[0121] DMEM without penicillin-streptomycin was added. The cells were then treated with 125 ng of mRNA per 18,000 cells to evaluate in vitro luciferase expression mediated by each LNP. DMEM alone served as the negative control, while LNPs formulated with MC3 were used as the positive control. For in vitro Vitamin D Receptor (VDR) corona study, luciferase mRNA LNPs were incubated with Recombinant Human Vitamin D Receptor (NovateinBio) at doses of 0, 0.1 , 0.25, 0.5, 0.75 and 1 pg of protein per pg of lipid for 15 min at 37°C with gentle shaking at 300 rpm. The LNP-treated cells were incubated at 37°C for 24 hours. Following incubation, 100 pL of luciferase assay substrate (Promega) was added to each well. The plate was shaken on a plate reader at 200 rpm in the dark for 10 minutes, and luminescence intensity was measured using the GloMax Explorer plate reader (Promega, USA).

[0122] Cryo-TEM Sample Preparation and Imaging

[0123] Three microliters of LNPs in a buffer solution were applied to a lacey copper grid coated with a continuous carbon film. The excess sample was carefully blotted away using the Gatan Cryo Plunge III. The grid was then mounted on a Gatan 626 single tilt cryo-holder and inserted into the TEM column. The specimen and holder tip were cooled with liquid nitrogen, maintaining the temperature throughout the transfer into the microscope and during imaging. Imaging was performed on a JEOL 2100 FEG microscope using the minimum dose method, essential for minimizing sample damage from the electron beam. The microscope was operated at 200 kV, with magnifications ranging from 10,000* to 60,000* to assess particle size and distribution. All images were recorded on a Gatan 2K * 2K UltraScan CCD camera.

[0124] Confocal Scanning Laser Microscopy (CLSM)

[0125] BxPC-3 cells were seeded in glass bottom dishes (Thermo Fisher, USA) and incubated for 24 h. The medium was then replaced with a culture medium containing DiO labelled LNPs (125 ng) for transfection for 2 hours. Briefly, LNPs encapsulating 125 ng of Flue mRNA LNPs labelled with DiO (0.5% molar ratio of LNPs) were added to the dishes. Cell nuclei and lysosomes were stained with Hoescht 33342 (Lumiprobe, USA) and Lysotracker Red (Invitrogen, USA). Images were obtained using a confocal scanning laser microscope (Nikon A1 , Japan) with a water immersion 63* objective lens. The imaging parameters were kept constant during the experiments.

[0126] H&E Staining 208034-0009-W001

[0127] Hematoxylin and eosin (H&E) staining on pancreas and liver tissues from C57BL / 6J mice were performed on formalin-fixed paraffin-embedded (FFPE) sections. Tissues were post-fixed in 10% formalin for 24-48 hours at room temperature (with the option to remain in formalin for up to a week) and then transferred directly to 70% ethanol. Histological processing was completed by HistoWiz Inc. (NY, USA) following their Standard Operating Procedure and fully automated workflow. Samples were embedded in paraffin and sectioned at 4 pm thickness. Formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene, rehydrated through graded ethanol, and rinsed in distilled water. Sections were stained with Hematoxylin for 5 minutes, rinsed in tap water, differentiated in acid alcohol, and treated with a bluing reagent. Eosin staining was applied for 1-2 minutes, followed by brief rinsing. Slides were then dehydrated through graded ethanol, cleared in xylene, and coverslipped with a compatible mounting medium. Whole slide scanning at 40x magnification was performed using an Aperio AT2 system (Leica Biosystems).

[0128] Immunofluorescence

[0129] Unstained slides of Ai14 mouse pancreas tissue samples post-treatment were obtained from HistoWiz Inc. (NY, USA). Sections, 5 pm thick, were deparaffinized in xylene, rehydrated through a graded ethanol series, and subjected to antigen retrieval in citrate buffer (pH 6.0) at 95°C for 20 minutes. After cooling to room temperature, the sections were permeabilized with 0.1 % Triton X-100 in PBS for 10 minutes and blocked with 5% normal goat serum in PBS for 1 hour. Immunofluorescent staining was performed using primary antibody against insulin (bio- techne, USA) followed by incubation with secondary antibody Alexa Fluor 488 (Thermo Fisher, USA) conjugated to fluorescent dyes. DAPI (1 pg / mL in PBS) was used for 10 minutes to stain nuclei. After washing with PBS, slides were mounted using an anti-fade mounting medium and covered with glass coverslips. Images were captured using a confocal scanning laser microscope (Nikon A1 , Japan) with an oil immersion 40* objective lens with filters for tdTomato (red fluorescence), insulin (green fluorescence), and DAPI (blue fluorescence).

[0130] Multiplex Analysis of Cytokines

[0131] This study used Luminex xMAP technology for multiplexed quantification of 10 Mouse cytokines, chemokines and growth factors. The multiplexing analysis was performed using the Luminex™ 200 system (Luminex, Austin, TX, USA) by Eve Technologies Corp. (Calgary, Alberta). Ten markers were simultaneously measured in the samples using Eve Technologies' Mouse Focused 10-Plex Discovery Assay® (MilliporeSigma, Burlington, Massachusetts, USA) according 208034-0009-W001 to the manufacturer's protocol. The 10-plex consisted of GM-CSF, IFNy, I L-1 p, IL-2, IL-4, IL-6, IL-10, IL-12p70, MCP-1 , and TNFa. Assay sensitivities of these markers range from 0.4 - 10.9 pg / mL for the 10-plex. Individual analyte sensitivity values are available in the MilliporeSigma MILLIPLEX® MAP protocol.

[0132] Animal Experiments

[0133] All animal procedures were conducted in accordance with guidelines and approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Las Vegas, Nevada (protocol #01218). Female and male C57BL / 6J mice (6-8 weeks old, approximately 20 g) and B6.Cg-Gt(ROSA)26Sortm14(CAG-tdTomato)Hze / J mice (6-8 weeks old, approximately 20 g) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Mice were injected with LNPs formulated with FLuc mRNA at a dose of 0.5 mg / kg via the lateral tail vein. For mechanism studies, female C57BL / 6J mice (6-8 weeks old, approximately 20 g) were intraperitoneally injected with 50 mg / kg of MeTC7 (MedChemExpress, USA). After 12 hours of administration of MeTC7, mice were intravenously injected via the lateral tail vein with FLuc mRNA LNPs at a dose of 0.5 mg / kg. A D-luciferin solution (30 mg / mL in 1 * PBS; PerkinElmer) was prepared, and mice were injected intraperitoneally with 130 L of this solution after 24 hours (for intravenous injections). After a 10-minute incubation, the mice were euthanized using CO2, and organs (liver, spleen, kidney, pancreas, heart, lung) were harvested and imaged with an in vivo imaging system (MS; PerkinElmer, Waltham, MA, USA). Luminescence flux was quantified using Living Image Software (PerkinElmer). For toxicity assessment, blood samples were collected via cardiac puncture 24 hours after treatment. Mice were then sacrificed by CO2 asphyxiation, and organs were collected. Blood samples were allowed to coagulate for 20 minutes at room temperature and centrifuged at 2000 g for 20 minutes at 4 °C to obtain high-quality of serum. Serum liver enzyme levels were measured by VRL Animal Health Diagnostics and cytokine panel was performed by Eve Technologies Corp. (Calgary, Alberta). For Ai14 mice experiments, fluorescence was quantified at an excitation / emission of 554 / 581 nm. Regions of interest (ROIs) of a constant size were placed around each organ's image, and total luminescence flux and radiant efficiency were reported as mean ± standard deviation.

[0134] Statistics

[0135] Statistical analysis of the results was performed by One-Way ANOVA followed by Bonferroni post-hoc analysis to compare multiple replicate means using Prism 10 (GraphPad). Differences were considered significant when p < 0.05. 208034-0009-W001

[0136] Traditional LNPs for mRNA delivery consist of four key components, each tailored to establish a stable lipid bilayer structure. These lipids include (1) ionizable lipids for complexing with mRNA with charge-altering properties, facilitating the release of mRNA cargo in the acidic endosomal environment; (2) helper lipids for bilayer structure reinforcement; (3) cholesterol for increasing structural integrity; and (4) PEG lipids for extending circulation in the bloodstream. To enhance organ-specific delivery of LNPs, we added endogenous vitamins as a fifth component in the formulation. The vitamins A, B2, D3, E, and K1 were chosen for their unique biological functions, which can complement the LN P core in different ways. Vitamin A (retinol) is well-known for its role in cell differentiation and immune modulation. Vitamin B2 (riboflavin), although water- soluble, was included because it plays a crucial role in cellular metabolism and energy production. Vitamin D3 (cholecalciferol) is essential for calcium homeostasis and immune function, specifically targeting tissues that express the VDR. This vitamin is necessary for maintaining - cell function, supporting insulin release, reducing [3-cell apoptosis, and promoting overall cell health. Vitamin E (tocopherol) possesses significant antioxidant properties, and vitamin K1 (phylloquinone) is crucial for blood coagulation and bone health.

[0137] To further diversify our ENDO LNP design, we developed a comprehensive formulation library using previously studied ionizable lipids (ILs), including benchmark SM-102, DLin-MC3- DMA (MC3), and C12-200, which are known to traffic to the liver. Additionally, we used THP1 , which our research group previously established as an effective mRNA delivery system. 3060i10 was included due to its previously demonstrated high transfection efficiency in numerous preclinical studies. For an efficient fifth component formulation development, it is crucial to partially replace the ionizable lipid without affecting encapsulation or endosomal escape. We started our study by using a benchmark formulation containing ionizable lipid, helper lipid, cholesterol, and DMG-PEG 2000 at a 35:16:46.5:2.5 molar ratio, which was then modified by partially replacing the ionizable lipid or cholesterol. The four different formulation ratios were chosen with an increasing vitamin substitution (5-15 molar ratio) to systematically investigate the effect of vitamin molar ratios on the performances of these LNPs. The detailed formulation ratios are shown in FIG. 1C and Table 1. Designing the formulation ratio is as significant as selecting the suitable fifth component in this study as it impacts the physicochemical parameters of LNP, including particle size, encapsulation efficiency (EE%), and surface charge, which are important for efficient mRNA delivery. These interactions can often significantly change with minor changes in the lipid- to-vitamin ratio, altering the stability of particles in circulation and their able to escape from endosomal. We formulated a library of 100 different LNP formulations by combining five different 208034-0009-W001

[0138] ILs, five vitamins, and four different formulation ratios (5 ILs * 5 Vitamins 4 Formulation = 100 LNPs) in a 96-well plate format using hand mixing. We used firefly luciferase (FLuc) mRNA as our reporter because its non-secretory nature allows direct imaging and quantification of transfection efficiency. The particle size and PDI of each LNP formulation were measured using dynamic light scattering (DLS), as shown in FIG. 2-3. The results indicate that the average diameter of most LNPs was within a window of 60 to 140 nm and PDIs smaller than 0.2, indicating a relatively narrow size distribution. The uniformity ensures efficient cellular uptake and successful endosomal escape, demonstrating that ENDO LNPs were structurally similar to conventional LNPs.

[0139] Table 1. LNP Formulation Ratios for ENDO LNP Library

[0140] Formula Ionizable Lipid DOPE Cholesterol PEG 5thComponent

[0141] F1 30 16 46.5 2.5 5

[0142] F2 25 16 46.5 2.5 10

[0143] F3 20 16 46.5 2.5 15

[0144] F4 35 16 41.5 2.5 5

[0145] We assessed the in vitro transfection efficiencies of all the LNPs in various cell lines, including human embryonic kidney cells (HEK293), human foreskin fibroblasts (HFF), human umbilical vein endothelial cells (HUVEC), mouse macrophage cells (RAW264.7), and human microglia cells (HMC3). These cell types represent a diverse range of tissues, including the kidney, skin, vascular endothelium, immune system, and brain. A comprehensive analysis of the transfection data revealed several key trends, indicating a significant improvement in transfection by ENDO LNPs compared with control MC3 and SM-102. Notably, LNPs incorporating retinol, riboflavin, and phylloquinone as fifth component exhibited significantly higher transfection efficiency in HEK293 cells compared to other ENDO LNPs and the control group (FIG. 4A). In contrast, LNPs containing cholecalciferol as the fifth component and those with THP1 as the ionizable lipid showed reduced transfection efficiency in HEK293 cells. In HFF cells, SM-102 LNPs incorporating retinol, riboflavin, and tocopherol as fifth component, as well as 3060110 LNPs with retinol, riboflavin, and cholecalciferol, achieved transfection levels several-fold higher than control LNPs and other ENDO LNPs (FIG. 4B). Furthermore, formulations containing MC3 and THP1 as ionizable lipids exhibited lower transfection efficiency in HFF cells. In HUVEC cells, which form the inner lining of blood vessels, C12-200 LNPs with retinol and tocopherol, 3060i10- based LNPs with tocopherol, and SM-102 and MC3 LNPs containing cholecalciferol 208034-0009-W001 demonstrated significantly higher transfection than control LNPs and other ENDO LNPs (FIG. 4C). THP1 LNPs showed poor transfection in HUVEC cells. RAW264.7 cells, which resemble macrophages, displayed significantly higher transfection with C12-200-based LNPs containing retinol. Notably, SM- 102- based LNPs incorporating tocopherol achieved the highest transfection levels in these cells (FIG. 4D), likely due to enhanced cellular uptake and intracellular processing of the LNPs. Other LNP formulations showed limited transfection in RAW264.7 cells. In HMC3 microglial cells, C12-200 LNPs containing retinol, riboflavin, and phylloquinone exhibited several- fold higher transfection efficiency than controls (FIG. 4E). Similarly, SM-102 LNPs containing tocopherol and cholecalciferol, as well as MC3 LNPs with riboflavin and 3060i10 ionizable lipids combined with retinol and riboflavin, demonstrated enhanced transfection. However, LNPs formulated with THP1 as the ionizable lipid showed low transfection in HMC3 cells. Further, all the traditional four-component formulations of C12-200, SM-102, 3060i10, THP1 , and MC3 were tested across all the cell lines, and none exhibited superior transfection efficiency compared to the highlighted formulations in our study (FIG. 5). Hence, our findings highlight that incorporating the vitamins as a fifth component into the ENDO LNPs can greatly enhance transfection efficacy compared with traditional four-component LNPs, providing a rationale for tissue-specific delivery.

[0146] Next, we evaluated the in vivo delivery efficacy of the ENDO LNPs using comprehensive batch analysis. This approach accelerated our screening process significantly and resulted in a remarkable reduction in the number of animals, as well as time and cost. In this study, we categorized 100 LNPs into four groups based on their formulation ratios (F1, F2, F3, and F4). Each group, containing 25 formulations corresponding to its respective formulation ratio, was administered to C57BL / 6 mice via intravenous injection at a dosage of 0.5 mg / kg, and protein expression was assessed in major organs using an MS imaging system. Formulations F1 and F4 primarily showed transfection in the liver and, with additional expression in the spleen, intestines, and pancreas. Notably, formulations F2 and F3 demonstrated significant redirection to extrahepatic space, with F2 showing selective protein expression in the pancreas and minimal expression in other organs, including the spleen and liver (FIG. 6A). Extrahepatic formulations, F2 and F3, were then batched based on ionizable lipids (THP1, C12-200, SM-102, MC3, and 3060110) in five groups, allowing for a total administration of 10 LNPs per injection at the same dose. Remarkably, C12-200 batch showed selective transfection in the pancreas, whereas the other ILs resulted in broader expression across various organs (FIG. 6B). This highlights the significance of selecting the appropriate IL to achieve targeted mRNA delivery. Further, C12-200 was batched based on different vitamins (retinol, riboflavin, cholecalciferol, tocopherol, and phylloquinone). Among these, cholecalciferol exhibited selective mRNA transfection and protein 208034-0009-W001 expression in the pancreas, highlighting the potential of ENDO LNPs for more precise mRNA delivery (FIG. 6C). Throughout each phase of the screening process, SM-102 and MC3 were used as control LNPs with major accumulation in the liver.

[0147] To further validate our findings from the batch screening, we formulated C12-200 cholecalciferol F2 (C-CholF2) and C12-200 cholecalciferol F3 (C-CholF3) LNPs using a microfluidic device. Previous studies have shown that LNPs formulated via hand mixing and microfluidic mixing exhibit no significant differences in transfection, both in vitro and in vivo. Microfluidic mixing allows improved reproducibility by accurately mixing aqueous and ethanol phases, resulting in highly reproducible LNPs with desired physicochemical properties essential for clinical translation and large-scale production. The measurement of particle size and EE% of these LNPs indicated that both C-CholF2 and CholF3 had similar sizes and notably high EE%, demonstrating effective mRNA encapsulation (FIG. 7A). To validate the efficacy of these ENDO formulations in the pancreas, we intravenously injected each LNP at 0.5 mg / kg in C57BL / 6 mice, along with the C12-200 control. The results demonstrated that both C-CholF3 and CholF2 exhibited selective expression in the pancreas, with an average total flux of 1.04 x 108for C- CholF3 compared to 2.29 107for CholF2, indicating a 4.5-fold higher efficacy (FIG. 7B and FIG. 8). Thus, C-CholF3 can selectively target the pancreas with significantly higher efficiency, demonstrating a complete redirection of liver-targeting C12-200 LNPs with cholecalciferol (FIG. 7C). The cryogenic electron microscopy (cryo-EM) image of C-CholF3 ENDO LNPs showed a spherical shape with a multi-lamellar shell surrounding an amorphous core (FIG. 7D and FIG. 9). To assess the dose-dependency of C-CholF3 and understand whether the protein expression in the pancreas could be regulated, we intravenously administered 0.25 mg / kg, 0.5 mg / kg, and 1 mg / kg of C-CholF3 LNPs to C57BL / 6 mice and observed a distinguishable dose-dependent improvement in transfection efficiency (FIG. 10). This highlights the importance of optimizing dosing to maximize the efficiency of LNP-mediated mRNA delivery along with balancing safety at higher doses. These findings will be crucial in determining the therapeutic window and the dosing strategies of future clinical applications.

[0148] Next, we investigated the in vivo biocompatibility of C-CholF3. H&E staining, both after 24 h and 48 h, showed no morphological damage or inflammatory response in pancreatic tissues treated with C-CholF3 compared to the PBS treated control (FIG. 11A, FIG. 12-13). The islet cells remained intact, and there was no evidence of necrosis or degeneration. Additionally, the liver tissues were examined with H&E staining after 48h and showed no significant change compared to the control. These findings highlight the safety and tolerability of C-CholF3 and its potential for future clinical applications. In addition to histopathological examination, the overall 208034-0009-W001 safety profile of C-CholF3 LNPs was studied by continuously monitoring body weight and hematological parameters. Over 21 days, no significant decrease in body weight was observed in mice (FIG. 14). The levels of liver enzymes such as ALT, AST, and alkaline phosphatase were within the normal range, with no renal toxicity observed based on the levels of BUN and CREA (FIG. 11 B and FIG. 15). It is known that different LNPs can elicit varied cytokine responses depending on factors such as lipid composition, nanoparticle size, and surface properties, leading to differential activation of immune pathways. Proinflammatory cytokines, such as IL-1 p, IL-6, and TNF-a, were measured 24 hours after injection and found to be comparable to PBS control, further confirming the low immunogenicity of the LNP (FIG. 11C). Other immune biomarkers, including GM-CSF, IFNy, IL-2, IL-4, IL-10, I L-12p70, and MCP-1 , also showed comparable levels to the control, indicating minimal systemic inflammatory response that is ideal for repeat dosing of LNPs (FIG. 16). These findings highlight that C-CholF3 ENDO LNPs exhibit significantly higher biocompatibility and reduced toxicity compared to traditional C12-200 LNPs. This is likely due to the replacement of a substantial portion of C12-200 ionizable lipid in the standard four-component formulation with endogenous cholecalciferol, a naturally occurring compound, thereby improving safety and biocompatibility.

[0149] To ensure robustness and the stability of C-CholF3 LNPs for clinical applications, we stored them at both 4°C and -20°C for various durations (1 , 3, 7, and 21 days) and evaluated changes in their physiochemical properties and mRNA delivery efficacy. Throughout all storage conditions, the LNPs exhibited minimal changes in physiochemical properties, with parameters such as particle size, polydispersity index (PDI), ^-potential, and EE% being consistent. Notably, the LNPs demonstrated robust FLuc expression even after 21 days of storage without any cryoprotectants, indicating exceptional stability (FIG. 17). This suggests that C-CholF3 LNPs can maintain their mRNA delivery potential over extended storage periods and can be adopted for long-term storage and transport, making them suitable for clinical and commercial use. We also assessed the in vivo stability and kinetics of C-CholF3 LNPs to understand and determine their potential for extended release. For this, we administered the nanoparticles intravenously into C57BL / 6 mice and monitored the bioluminescence signal over 72 hours. We observed that the bioluminescence intensity remained robust for up to 72 hours post-injection, indicating prolonged stability and expression of the delivered mRNA (FIG. 18). The higher stability of C-CholF3 LNPs could be advantageous for applications requiring prolonged gene expression in vivo.

[0150] Organ tropism has long been understood to be influenced by the apparent pKa of LNPs, with lower pKa values typically targeting the spleen and higher values favoring lung or liver delivery. However, pancreas-targeting LNPs had not been previously characterized. Using TNS 208034-0009-W001 assay, we found that the apparent pKa of the pancreas-targeting C-CholF3 LNPs is 7.31 , compared to a pKa of 6.71 for the liver-targeting C12-200 LNPs (FIG. 19). To expand the therapeutic applicability of C-CholF3 LNPs beyond mRNA delivery, we evaluated their ability to deliver other clinically relevant nucleic acid cargoes, such as plasmid DNA (pDNA) and circular mRNA (circ mRNA), which are increasingly being explored for long-term expression and enhanced molecular stability, respectively. Following intravenous administration of C-CholF3 LNPs containing either circ mRNA or pDNA, we observed the average total flux values at 8.41 * 107for circ mRNA and 1 x 108for pDNA with over 99% selectivity for both (FIG. 20), highlighting broad compatibility of the formulation to different nucleic acids. The ability to deliver multiple forms of genetic material is essential for expanding the therapeutic potential of pancreatic gene delivery. Plasmid DNA allows long-term expression of therapeutic genes, and circular mRNA offers enhanced stability with reduced immunogenicity compared with linear mRNA. Overall, our findings indicate that C-CholF3 LNPs are a safe and highly effective method for delivering pancreas-targeted nucleic acid therapeutics with significant implications for pancreatic cancer and diabetes therapies.

[0151] We next aim to elucidate the mechanism by which C-CholF3 ENDO LNPs enable pancreas selective RNA delivery. To achieve this, we first investigated their cellular uptake and trafficking in BxPC-3 human pancreatic cancer cells. Using confocal microscopy, we monitored the internalization and intracellular localization of DiO-labeled C-CholF3 and MC3 LNPs. At 2 hours post-treatment, C-CholF3 LNPs exhibited pronounced colocalization with LysoTracker- labeled endo / lysosomal vesicles, indicating effective internalization and endosomal trafficking (FIG. 21A, and FIG. 22-24). Notably, BxPC-3 cells treated with C-CholF3 displayed significantly higher DiO fluorescence compared to MC3-treated cells, suggesting enhanced cellular uptake potentially due to receptor-mediated internalization pathways. Many LNPs have shown to achieve liver and spleen tropism by forming an ApoE-rich protein corona and a [32-glycoprotein I (|32-GPI)- rich protein corona, respectively. Given that C-CholF3 contains cholecalciferol as the fifth component in the LNP formulation, we hypothesized that interaction with the VDR may lead to selective uptake by pancreatic cells. Furthermore, soluble VDR might form a corona on the C- CholF3 surface, facilitating receptor-mediated internalization. To test this hypothesis, we preincubated C-CholF3 LNPs with increasing levels (0-1 pg) of recombinant human VDR protein prior to transfection into BxPC-3 cells (FIG. 21 B). We observed a dose-dependent increase in luciferase expression, indicating that binding to VDR enhanced the delivery and functional expression of mRNA (FIG. 21 C). Notably, CholF3 LNPs coated with 1ug VDR resulted in a 3.7- fold improvement in protein expression. This suggests that VDR binding on the nanoparticle 208034-0009-W001 surface promotes more efficient cellular uptake and internalization through receptor-mediated endocytosis.

[0152] We then investigated the proposed mechanism underlying pancreas tropism in vivo. We employed MeTC7 for this purpose, which is a potent and selective antagonist of VDR. By preadministering MeTC7 before LNP treatment, we aimed to block the VDR from facilitating tissue- selective delivery. This pharmacological strategy enables an acute disruption of VDR-mediated processes without requiring genetic knockout models, offering valuable mechanistic insight into the role of VDR-dependent pancreatic delivery of C-CholF3 LNPs. Twelve hours prior to intravenous injection of C-CholF3 ENDO LNPs encapsulating Flue mRNA (0.5 mg / kg), we pretreated C57BL / 6 mice intraperitoneally with MeTC7 (50 mg / kg) or PBS as control (FIG. 21 D). IVIS imaging 24 hours post-injection revealed robust luciferase expression in the pancreas of PBS-treated mice, confirming selective tropism of C-CholF3 (FIG. 21 E and FIG. 25). In contrast, mice pre-treated with MeTC7 displayed a complete loss of pancreatic signal, supporting the requirement of functional VDR for tissue-selective delivery. Moreover, MC3 LNPs delivered to MeTC7-treated mice continued to exhibit strong hepatic protein expression, consistent with the known mechanism of Apo E- mediated liver targeting. These finding underscore the mechanistic difference between liver-targeting LNPs and pancreas-targeting LNPs, emphasizing the critical role of VDR in C-CholF3-mediated delivery. To further investigate the role of VDR and assess whether its potential application in targeting could extend beyond cholecalciferol to other VDR binding molecules, we replaced cholecalciferol with MeTC7 as a fifth component in pancreas targeting formulation and evaluated its effect on pancreas tropism in vivo. MeTC7 LNPs were intravenously administered at a dose of 0.5 mg / kg, and 24 hours post-injection, we observed selective and robust luciferase expression in the pancreas (FIG. 21 F and FIG. 26). These results support a novel VDR-mediated delivery mechanism. By exploiting the tissue-resident or overexpressed receptors through rational lipid design, such as incorporating endogenous ligand cholecalciferol, it is possible to achieve organ-selective gene delivery with high specificity. This receptor-ligand matching approach paves the way for rational designs of LNPs to other extrahepatic tissues by aligning lipid ligands with tissue-specific receptors.

[0153] Having demonstrated that C-CholF3 can deliver mRNA to the pancreas, we next utilized the Ai14 mouse model to assess the tissue-specific gene-editing capability of C-CholF3 LNPs. The Ai14 mice are genetically engineered and contain a construct designed for Cre-mediated gene editing, featuring a LoxP-stop-LoxP cassette downstream of the CAG promoter. After successful delivery of Cre recombinase, the stop cassette is excised, activating tdTomato fluorescence in the targeted cells. Thus, the system simplifies the identification and quantification 208034-0009-W001 of the gene-edited cells, as the cells with Cre mRNA expression exhibit characteristic red tdTomato fluorescence in a tissue-specific manner (FIG. 27A). We formulated the C-CholF3 LNPs with Cre recombinase mRNA and intravenously injected them into the Ai14 mice at 1.5 mg / kg. After 120 h, fluorescence imaging revealed robust tdTomato expression in the pancreas (FIG. 27B) with more than 99% selectivity (FIG. 27C and FIG. 28). Minimal fluorescence was observed in other tissues, such as the liver, where LNP uptake and Cre expression were not statistically significant compared to those in the pancreas (FIG. 28). This suggests that the formulation may be applicable to other animal models. Following IVIS detection of tdTomato, we performed immunofluorescence (IF) analysis to understand which cells were undergoing tissue-specific gene editing. Staining with insulin antibodies identified tdTomato fluorescence within insulinpositive cells, indicating effective gene editing in pancreatic p cells by C-CholF3 ENDO LNPs (FIG. 27D, and FIG. 29-30). However, this delivery is not exclusive to p-cells. These results show that this delivery system can modulate gene expression in major pancreatic cell types, which makes it promising for targeted treatments in pancreatic diseases.

[0154] To better evaluate the clinical translatability of C-CholF3 LNPs, we compared their safety profile with clinically approved MC3 formulation (Onpattro). We assessed in vivo transfection efficiency and toxicity profiles of C-CholF3 and MC3 at 4 and 24 hours post-injection. IVIS imaging at 4 hours (FIG. 31A) demonstrated early pancreatic accumulation of C-CholF3, with an average total flux of 3.52 x 106, and no redistribution following liver clearance. In contrast, MC3 predominantly accumulated in the liver, exhibiting a higher average total flux of 2.26 x 107. At 24 hours, IVIS images (FIG. 31 B) confirmed enhanced pancreatic transfection with C-CholF3 (average total flux of 3.47 x 107) compared to MC3 (average total flux of 3.24 x 107). Safety evaluations indicated that C-CholF3 exhibited lower serum levels of liver enzymes ALT and AST (FIG. 32A) compared to MC3, similar kidney function parameters were observed for both C- CholF3 and MC3 (FIG. 32B), and a more favorable cytokine profile (FIG. 32C). Notably, IL-6 levels were significantly lower with C-CholF3, whereas IL-1 f3 levels were higher than those observed with MC3, overall supporting a superior safety profile for C-CholF3.

[0155] In summary, we report that the addition of endogenous cholecalciferol as a fifth component successfully redirects traditional four-component C12-200 LNPs from the liver to the pancreas following intravenous administration. Using a comprehensive screening approach, we identified C-CholF3 LNPs containing cholecalciferol as a fifth component. This formulation demonstrated robust protein expression in the pancreas with over 99% selectivity and significantly reduced toxicity. We propose that the mechanism underlying pancreas tropism in vivo is driven by an endogenous targeting mechanism involving VDR. Additionally, C-CholF3 facilitates selective 208034-0009-W001 pancreatic delivery of both plasmid DNA and circular mRNA, showcasing its versatility and therapeutic promise. Furthermore, C-CholF3 showed robust pancreas-specific tdTomato expression, especially in pancreatic p cells of the Ai14 transgenic mouse model, highlighting the effectiveness of C-CholF3 LNPs in gene editing. These findings indicate that formulations with the appropriate endogenous fifth component could be utilized to redirect therapies to extrahepatic space with minimal toxicity, potentially advancing the development of gene therapy or personalized medicine for pancreatic diseases with the capability of repeat administration.

[0156] Example 2

[0157] Intramuscular Delivery of ENDO LNPs

[0158] By organizing the 100 LN P formulations into four distinct groups based on their formulation ratios (F1, F2, F3, and F4), an efficient high-throughput in vivo screening was conducted using intramuscular administration. Each group, containing 25 formulations, was administered intramuscularly to C57BL / 6 mice at a dosage of 0.5 mg / kg. Protein expression was then evaluated using an IX / IS imaging system.

[0159] The results revealed that formulations in the F1 and F2 groups exhibited the highest levels of mRNA transfection at the injection site (FIG. 33A). Specifically, LNPs from the F1 group demonstrated an average total flux of 1.54 x 109, while those from the F2 group yielded 2.33 x 109total flux. Given the superior transfection observed in these groups, the LNP composition was further optimized by subdividing the F1 and F2 batches based on their ionizable lipid (IL) components, including THP1 , C12-200, SM-102, MC3, and 3060i10. This second round of screening involved administering ten LNP formulations per injection for each IL group. Remarkably, the C12-200-based LNPs demonstrated the highest levels of transfection in muscle tissue, with an average total flux of 4.13 x io9(FIG. 33B). To refine the LNP design even further, a batch analysis was then performed based on the fifth component, the endogenous vitamins. C12-200-based LNPs were subdivided into five groups, each incorporating a different vitamin component: retinol (Vitamin A), riboflavin (Vitamin B2), cholecalciferol (Vitamin D3), tocopherol (Vitamin E), and phylloquinone (Vitamin K). Among these, retinol and tocopherol formulations showed the highest mRNA transfection efficiency in the muscle tissue. The retinol-based LNPs exhibited an average total flux of 1.60 x 109, while the tocopherol-based LNPs showed 6.62 x 108(FIG. 33C).

[0160] These results further highlight the capability of ENDO LNPs to enable precise, tissuespecific mRNA delivery. Throughout each phase of the screening process, the benchmark ionizable lipids SM-102 and MC3 served as controls for intramuscular mRNA delivery. Consistent 208034-0009-W001 with previous studies, both control LNPs exhibited moderate mRNA transfection but were outperformed by the C12-200-based formulations, thus confirming the superior performance of the optimized ENDO LNPs.

[0161] Following the initial screening, four top-performing formulations were identified: C-RetF1 , C-TocF1 , C-RetF2, and C-TocF2. To further validate these hits and assess their in vivo delivery efficacy, each formulation was administered intramuscularly at a dose of 0.5 mg / kg per animal. As controls, the conventional four-component C12-200 LNP was included, given that C12-200 was the lead ionizable lipid in the top-performing formulations, as well as the standard SM-102 LNP, which has been widely used in mRNA COVID-19 vaccines. This allowed for directly comparing the efficacy of the ENDO LNPs to established systems.

[0162] The in vivo validation revealed that C-RetF1 exhibited the highest level of transfection compared to the other formulations and controls (FIG. 34A-B). Specifically, C-RetF1 achieved an average total flux of 2.24 x 109, which represents a remarkable 20-fold increase in transfection efficiency compared to the conventional four-component C12-200 LNPs, which showed an average total flux of 1.13 x 108. This significant improvement highlights the potential of the retinol- based ENDO LNPs to enhance mRNA delivery for therapeutic applications.

Claims

208034-0009-W001CLAIMSWhat is claimed:

1. A lipid nanoparticle composition comprising: one or more ionizable lipids; one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder.

2. The composition of claim 1 , wherein the composition comprises about 10 mol% to about 15 mol% vitamin D or a vitamin D receptor binder.

3. The composition of claim 1, wherein the vitamin D is vitamin D3 (cholecalciferol).

4. The composition of claim 1 , wherein the vitamin D receptor binder is a selective antagonist of the vitamin D receptor.

5. The composition of claim 1 , wherein the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof.

6. The composition of claim 1 , wherein the one or more non-ionizable lipids comprise phospholipids.

7. The composition of claim 6, wherein the phospholipids are selected from the group consisting of 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), and combinations thereof.

8. The composition of claim 1 , wherein the one or more PEG-modified lipids are selected from the group consisting of 1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol- 2000 (DMG-PEG 2000), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-208034-0009-W001[amino(polyethylene glycol) (DSPE-PEG), 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine with conjugated methoxyl polyethylene glycol) (mPEG-DSPE), 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)- 2000] (14:0 PEG2000 PE), and combinations thereof.

9. The composition of claim 1 , wherein the composition comprises: about 20 mol% to about 35 mol% ionizable lipids; about 15 mol% to about 17.5 mol% non-ionizable lipids; about 1 mol% to about 4 mol% PEG-modified lipids; and about 40 mol% to about 48 mol% cholesterol or a derivative thereof.

10. The composition of claim 1 , further comprising a therapeutic agent encapsulated in the lipid nanoparticle.

11. The composition of claim 10, wherein the therapeutic agent comprises one or more nucleic acid molecules.

12. The composition of claim 11 , wherein the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof.

13. The composition of claim 11 , wherein the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof.

14. The composition of claim 1 , wherein the lipid nanoparticle has a diameter size of about 50 nm to about 200 nm.

15. The composition of claim 1 , wherein the lipid nanoparticle has a polydispersity index (PDI) of about 0.05 to about 0.3.

16. A method of delivering a lipid nanoparticle composition to a pancreas of a subject, the method comprising: administering to a subject a lipid nanoparticle composition comprising: one or more ionizable lipids;208034-0009-W001 one or more non-ionizable lipids; one or more PEG-modified lipids; cholesterol or a derivative thereof; and about 5 mole percent (mol%) to about 25 mol% vitamin A, vitamin B, vitamin D, vitamin E, or vitamin K; or about 5 mol% to about 25 mol% a vitamin A receptor binder, a vitamin B receptor binder, a vitamin D receptor binder, a vitamin E receptor binder, or a vitamin K receptor binder.

17. The method of claim 16, wherein the lipid nanoparticle composition is administered to the subject by intravenous, intraperitoneal, or intramuscular injection.

18. The method of claim 16, wherein the vitamin D is vitamin D3 (cholecalciferol).

19. The method of claim 16, wherein the vitamin D receptor binder is a selective antagonist of the vitamin D receptor.

20. The method of claim 16, wherein the one or more ionizable lipids comprise cationic lipids selected from the group consisting of C12-200, SM-102, and combinations thereof.

21. The method of claim 16, wherein at least 95% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration.

22. The method of claim 16, wherein at least 99% of the lipid nanoparticle composition is delivered to the pancreas of the subject following administration.

23. The method of claim 16, wherein a portion of the lipid nanoparticle composition is delivered to one or more non-pancreas organs in the subject following administration.

24. The method of claim 23, wherein the non-pancreas organs comprise one or more muscles, lymph nodes, or a combination thereof in the subject.

25. The method of claim 16, wherein the composition further comprises a therapeutic agent encapsulated in the lipid nanoparticle.208034-0009-W00126. The method of claim 25, wherein the therapeutic agent comprises one or more nucleic acid molecules.

27. The method of claim 26, wherein the one or more nucleic acid molecules comprise DNA, RNA, mRNA, or a combination thereof.

28. The method of claim 26, wherein the one or more nucleic acid molecules comprise one or more plasmid DNA, circular mRNA, Cas9 mRNA, guide RNA, or a combination thereof.

29. The method of claim 16, wherein the method treats, ameliorates, or inhibits the progress of a pancreatic disease in the subject.

30. The method of claim 29, wherein the pancreatic disease is selected from the group consisting of diabetes, pancreatic cancer, pancreatitis, and combinations thereof.

31. The method of claim 16, wherein the method results in gene editing, protein replacement, or a combination thereof in the pancreas.

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