Targeted nanomedicine for treating diabetes

Abstract

This disclosure relates to compositions and methods for treating diabetes, including, for example, Type 1 diabetes (TID).

Description

MBHB Ref. No.24-0108-WO TARGETED NANOMEDICINE FOR TREATING DIABETES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63 / 555,832, filed February 20, 2024, which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under HL161244 awarded by the National Institutes of Health. The government has certain rights in the invention. SEQUENCE LISTING

[0003] A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on February 17, 2025, having the file name “24- 0108-WO.xml” and is 8.22 kb in size. BACKGROUND OF THE DISCLOSURE Field of Invention

[0004] This disclosure relates to compositions and methods for treating diabetes, including, for example, Type 1 diabetes (T1D). Technical Background

[0005] Type 1 diabetes (T1D) research has long focused on the role of T-cells in mediating cellular autoimmunity with subsequent loss of -cell mass. Although therapies that primarily target the immune system have shown promise in preventing or stabilizing disease progression, clinical trials suggest limited efficacy which emphasizes the need to consider the impact of non- immune cells in disease pathogenesis. Recent studies propose that -cells may initiate or propagate T1D pathogenesis by producing aberrant proteins that serve as neoantigens for subsequent immune activation. The discovery that -cells may actively promote T1D progression presents new opportunities for targeted-cell drug development. -cell-centric therapies could help reduce -cell stress, mitigate neoantigen production, and preserve -cell function, potentially delaying diabetes onset with minimal off-target effects. Importantly, this goal cannotMBHB Ref. No.24-0108-WO be achieved with systemic drug delivery unless the targets are -cell-specific. Therefore, an unmet need in the field is to develop technologies that deliver therapeutics specifically to -cells and, in doing so, alleviate the underlying defects that promote T1D.

[0006] In recent years, lipid nanoparticles (LNPs) have emerged as promising vehicles for cell-specific drug delivery and have been safely and effectively used to deliver immune- sensitizing cargo in the form of COVID-19 vaccines. Nanoparticles can not only encapsulate various therapeutic cargo (e.g. mRNA, bioactive peptides, and CRISPR products) but also undergo modification to increase specificity on targeted cells of interest. Although this cell- targeting technology has been successfully used to modulate immune responses and combat cancer, it has yet to be leveraged to target -cells in the context of diabetes. Therefore, targeted nanomedicine approaches with tremendous potential to treat T1D were developed as described herein. BRIEF SUMMARY OF THE DISCLOSURE

[0007] This disclosure describes compositions and methods for treating diabetes, including, for example, Type 1 diabetes (T1D).

[0008] In a first aspect, the present disclosure provides a targeted nanoparticle, comprising a lipid; a -cell specific targeting peptide; and a nucleic acid molecule.

[0009] In some embodiments of the first aspect, the lipid comprises a lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14), cholesterol, polyethylene glycol 2000 (PEG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), and dioleoylphospha-tidylethanolamine (DOPE).

[0010] In some embodiments of the first aspect, the targeted nanoparticle comprises a molar ratio of the lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14) to DSPE-PEG of about 2:1 to about 15:1, cholesterol to DSPE-PEG2k is about 15:1 to about 40:1, and DOPE to DSPE-PEG is about 15:1 to about 30:1.

[0011] In some embodiments, the PEG domain comprises PEG having an average molecular weight of about 1,000 to about 100,000 Daltons.

[0012] In some embodiments of the first aspect, the lipid comprises a lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14), cholesterol, 1,2-Distearoyl-sn-glycero-3-MBHB Ref. No.24-0108-WO phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), and dioleoylphospha- tidylethanolamine (DOPE), and a nucleic acid molecule encapsulated by the lipid.

[0013] In some embodiments of the first aspect, the lipid encapsulates about 1 M to about 100 M of the nucleic acid molecule.

[0014] In some embodiments of the first aspect, the targeting peptide is a glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), or an FXYD domain containing ion transportregulator 2 a (FXYD2 a).

[0015] In some embodiments of the first aspect, the targeting peptide is the glucagon-like peptide-1 (GLP-1).

[0016] In some embodiments of the first aspect, the targeted nanoparticle comprises about 3×10-10μM to about 2×10-8μM of the -cell specific targeting peptide.

[0017] In some embodiments of the first aspect, the nucleic acid molecule comprises an RNA molecule encoding an immune-regulatory protein.

[0018] In some embodiments of the first aspect, the immune regulatory protein is an immune checkpoint protein, a cytokine, or -cell transcriptional factor.

[0019] In some embodiments of the first aspect, the RNA molecule is an mRNA molecule.

[0020] In some embodiments of the first aspect, the mRNA molecule is an immune- regulatory mRNA or -cell transcriptional factors.

[0021] In some embodiments of the first aspect, the immune checkpoint protein is PD-L1.

[0022] In some embodiments of the first aspect, the targeted nanoparticle is configured to deliver the nucleic acid molecule to the pancreas.

[0023] In a second aspect, the present disclosure provides a method of treating diabetes in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, and a nucleic acid encoding PD-L1 encapsulated within the lipid, wherein the targeted nanoparticle is preferentially targeted to a dysfunctional pancreatic cell, thereby modifying diabetes disease progression, and preventing or reversing disease occurrence.

[0024] In some embodiments of the second aspect, the diabetes is Type 1 diabetes (T1D).MBHB Ref. No.24-0108-WO

[0025] In some embodiments of the second aspect, the dysfunctional pancreatic cell is a - cell.

[0026] In a third aspect, the present disclosure provides a method of treating, preventing, or delaying symptoms of pre-diabetes in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, and a nucleic acid encoding PD-L1 encapsulated within the lipid, wherein the subject has stage 1 or stage 2 pre-diabetes, and wherein the targeted nanoparticle is preferentially targeted to a dysfunctional pancreatic cell; delivering the nucleic acid encoding PD-L1 to -cells, promoting -cell survival, restoring functional -cells, and reducing insulitis, thereby treating, preventing, or delaying symptoms of pre-diabetes in the subject.

[0027] In a fourth aspect, the present disclosure provides a pharmaceutical composition, comprising a therapeutically effective amount of targeted nanoparticles comprising a lipid, a - cell specific targeting peptide, and a nucleic acid molecule encoding a therapeutic peptide encapsulated within the lipid, and a pharmaceutically acceptable carrier, solvent, adjuvant, and / or diluent.

[0028] In some embodiments of the fourth aspect, the lipid comprises a lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14), cholesterol, polyethylene glycol 2000 (PEG), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), and dioleoylphospha-tidylethanolamine (DOPE).

[0029] In some embodiments of the fourth aspect, the DSPE-PEG comprises a PEG domain comprising PEG having an average molecular weight of about 1,000 to about 100,000 Daltons.

[0030] In some embodiments of the fourth aspect, the -cell specific targeting peptide comprises a glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), and / or anFXYD domain containing ion transport regulator 2 a (FXYD2 a).

[0031] In some embodiments of the fourth aspect, the therapeutic peptide comprises PD-L1, IL-12, IL-23, PAX6 and / or MAF-B.

[0032] In some embodiments of the fourth aspect, the pharmaceutical composition is formulated for inhalation, insufflation, oral, intravenous, topical, ocular, buccal, systemic, nasal, injection, transdermal, rectal, or vaginal administration.MBHB Ref. No.24-0108-WO

[0033] In some embodiments of the fourth aspect, the pharmaceutical composition is formulated for inhalation, intraperitoneal or intravenous administration.

[0034] In a fifth aspect, the present disclosure provides a pharmaceutical composition for pancreatic delivery of an RNA molecule comprising a targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, a nucleic acid molecule encoding a therapeutic peptide encapsulated within the lipid, and a pharmaceutically acceptable carrier, wherein the composition is formulated such that once administered to a subject, the targeted nanoparticle delivers the nucleic acid molecule to -cells in the pancreas.

[0035] These and other features and advantages of the present invention will be more fully understood from the following detailed description taken together with the accompanying claims. It is noted that the scope of the claims is defined by the recitations therein and not by the specific discussion of features and advantages set forth in the present description. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The accompanying drawings are included to provide a further understanding of the methods and compositions of the disclosure, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the disclosure, and together with the description serve to explain the principles and operation of the disclosure.

[0037] FIG.1 shows the circuit model of type 1 diabetes (T1D). Environmental insults, mediated through macrophages, trigger cell inflammation, generating neoantigens that trigger autoimmunity.

[0038] FIG.2 shows target receptors on cells, such as GLP-1R. GLP-1R is the most highly expressed and specific within cells.

[0039] FIG.3 shows the overall approach for the experiments.

[0040] FIG.4 shows development of LNPs to target pancreatic -cells to both mouse and human -cell populations with a therapeutic goal to test PD-L1 cargo in a pre-diabetic mouse model to delay T1D progression.

[0041] FIG.5 shows that -cells are key players in pre-T1D onset (Atkinson and Mirmira, Cell Metabolism, 2023). The right panel shows healthy beta cells versus beta cells alterations in pre-T1D.MBHB Ref. No.24-0108-WO

[0042] FIGS.6A-6D shows the identification and characterization of an LNP nanoparticle. Fig.6A shows a diagram of composition of a core C9-LNP. Figure 6B presents a transmission electron microscopy (TEM) image of C9-LNP loaded with KLF2 mRNA, revealing a monodisperse lipid nanoparticle with a diameter of approximately 70 nm under dry conditions. Figure 6C illustrates the hydrodynamic properties of KLF2 mRNA-encapsulated C9-LNP, showing a median diameter of approximately 120 nm in deionized (DI) water. Fig.6D shows a C9-LNP shows 98% mRNA encapsulation efficiency (top) and keeps consistent mRNA transfection efficiency by testing C9-LNPs from different synthetic batches (bottom).

[0043] FIGS.7A-7C show VCAM1-targeting LNPs (Fig.7A) characterized by transmission electron microscope (TEM) and dynamic light scattering (DLS), (Fig.7B) effectively delivered functional mScarlet mRNA to inflamed pulmonary microvascular endothelial cells in vitro, (Fig. 7C) delivered functional mScarlet mRNA to inflamed lung in vivo. (n=5, representative data / images shown).

[0044] FIG. 8 shows human EndoC- H1 cells and mouse MIN6 cells were exposed invitro to LNPs containing mRNA encoding the mScarlet protein. Shown are live-cell images of cells following 6 hrs of LNP exposure. Approximately 80% of cells in each case express mScarlet protein.

[0045] FIG. 9 shows LNP mediated delivery of HEK293 cells and EndoC- H1 cells withmScarlet mRNA cargo. Left panel depicts eGLP1-LNPs showing preferential transduction ofhuman EndoC- H1 cells compared to HEK293 cells in vitro by immunostaining. HEK293 andEndoC- H1 cells were cultured with LNPs that interact with GLP-1R and encapsulate mScarletmRNA. Cells were cultured for 4 or 18 hr with LNPs, washed, and recovered for 90 min prior to visualization for mScarlet. Right panel depicts DAPI overlap with mScarlet protein withinEndoC- H1 cells.

[0046] FIGS.10A-10C show that the characterization of eGLP1 LNPs. Fig.10A shows transmission electron microscopy (TEM) image of peptide functionalized LNPs. Fig.10B shows a bar graph of LNP size. Fig.10C shows a bar graph measuring LNP charge.

[0047] FIG.11 shows TEM analysis of untargeted (Control-LNPs) and targeted (eGLP1- LNPs) LNPs containing PD-L1-encoding mRNAs. Bottom left and right panels show the physical characteristics of untargeted and targeted LNPs, respectively. Note that the targeted LNPs had a slightly higher radius, as anticipated.MBHB Ref. No.24-0108-WO

[0048] FIGS. 12A-12C show C9-LNPs deliver Cd274 to MIN6 and EndoC- H1 cell linesand produce PD-L1 protein. The surface PD-L1 protein are expressed by untargeted C9-LNPs and eGLP1-C9 targeting LNPs on the surface of MIN6 mouse cells upon overnight exposure to LNPs (note that there is a slight preferential targeting efficiency by eGLP1-C9 LNPs). Fig.12A shows a representative FACS plots between conditions showing PD-L1 abundance after LNP delivery in MIN-6 cells. Fig. 12B shows bar plot quantification of FACS data (n=3). Fig.12Cshows a western blot of PD-L1 and Actin in EndoC- H1 cells after 1 or 3 days of LNP exposure.

[0049] FIGS.13A-13C show that eGLP1-LNPs show preferential transduction of intact mouse islets in vitro by flow cytometry. Fig.13A and Fig.13B show flow cytometry plots and Fig.13C shows quantification. Figs.13A and 13B show there is still a substantial transduction of cells which is an expected outcome because cells are present in the interior of the islet and this approach did not deliver NPs via vasculature (only diffusion)—and uptake by cells may represent background uptake.

[0050] FIGS.14A-14D shows in vitro LNP delivery with isolated mouse islets. Fig. 14A shows a schematic depicting LNP exposure on mouse islets. Briefly, after mouse islet isolation, islets were dispersed with accutase and then LNPs were added overnight in vitro and subsequently measured via FACS to measure fluorescence in various cell types. Fig. 14B shows Cy5 fluorescent abundance of Insulin (Ins) + and Glucagon (Gluc) – cells when examining multiple islet cell populations comparing between LNP treatment conditions. Fig.14C shows Cy5 fluorescent abundance when examining just cells (Ins+ and Gluc-) between conditions Fig.14D shows Cy5 fluorescent abundance of Gluc+ and Ins- cells when examining multiple islet cell population between conditions.

[0051] FIG.15 shows perifusion of human islets using the Biorep islet perifusion device.

[0052] FIGS.16A-16C show in vivo biodistribution of eGLP-1 LNPs with fluorescent cargo and the preferential targeting of eGLP1-LNPs to pancreas compared to control-NPs by IVIS (in vivo imaging system) with visualization of individual organs by measurement and quantitation of Cy5-tagged mRNA. Fig.16A shows in vivo experimental timeline with intraperitoneal delivery. Fig.16B shows representative images of all conditions utilizing in vivo fluorescent imaging system (IVIS) for 5 different conditions. Scale bar on left indicates radiant efficiency from lowest to highest gradient (black to yellow). Fig.16C shows quantification of pancreas radiant efficiency compared between all conditions. One-way ANOVA: ns= not significant; ** <0.005;MBHB Ref. No.24-0108-WO *** = 0.0001; **** < 0.0001. Red intensity in the figure indicates the levels of Cy5 targeting to pancreas.

[0053] FIG.17 shows a timeline to assess T1D Progression. Relating to Example 12 NOD mouse experiment from 6-25 weeks of age. First two weeks show bi-weekly IP injection (blue). Then the rest of the timeline follows (black arrow) out to endpoint of 25 weeks.

[0054] FIG.18 shows that in vitro LNP delivery is GLP-1R dependent. Left diagram shows dispersed mouse islets were treated with Exendin-4, a GLP-1R agonist, before overnight exposure with LNPs. Right graph shows Ins+ cells staining positive for Cy5 by flow cytometry. Numbers below show conditions with or without Exendin 4 in nM concentration.

[0055] FIGS.19A-19B show LNP targeting after using high dose of Streptozotocin (STZ). Fig.19A shows mouse mass and Fig.19B shows unfasted glucose between water injected mice and high dose streptozotocin (STZ) injected mice. STZ is a known chemical compound that kills cells. Mice are of C57BL / 6J background and the timeline for LNP treatment after STZ was after waiting 48 hours.

[0056] FIG.20 shows that using a high dose of STZ altered targeting enrichment. Furthermore, no specific enrichment of Cy5 expression was observed between untargeted & targeted LNPs. Quantification related to High Dose STZ injected images.

[0057] FIG.21 shows Cy5 quantification within primary human islets exposure to LNP conditions with Cy5-eGFP mRNA. Left panel shows Cy5 fluorescent abundance when examining cells (Ins+ and Gluc-) between untreatead controls, non-targeted LNPs, and targeted eGLP-1 LNPs. Right panel shows Cy5 fluorescent abundance within all Ins- cells when examining whole islet cell population between conditions.

[0058] FIG.22 shows immunofluorescence imaging of mouse islets focusing on PD-L1 expression in NOD mice after LNP mediated delivery of Cd274 mRNA cargo. DAPI in light grey, PD-L1 in dark grey, and INSULIN in medium grey.

[0059] FIGS.23A-23J show characteristics and in vitro efficacy of eGLP-LNPs. Fig. 23A shows transmission electron microscopy images of unconjugated (left panel) and eGLP (right panel) LNPs. Scale bars = 50 nm. Fig. 23B shows LNP size (nm). Fig.23C shows LNP size distribution (nm) of unconjugated (left panel) and eGLP (right panel) LNPs. Fig. 23D shows LNP zeta potential (mV). Fig.23E shows percent mRNA encapsulation efficiency. Fig. 23F is a graphical representation of a GLP-1R internalization assay measuring chemiluminescenceMBHB Ref. No.24-0108-WO activity. Fig.23G shows normalized expression of GLP-1R internalization. Concentrations were as follows: Exendin-4 (100 nM), eGLP-1 (40 nM), unconjugated LNP (50 ng), eGLP-LNP (50 ng). Fig.23H shows percent of dissociated mouse islets measuring Cy5 positive and insulin (INS) positive expression. Fig.23I shows percent of dissociated mouse islets measuring Cy5 positive and glucagon (GCG) positive expression. Fig.23J shows percent of dissociated mouse islets measuring Cy5 positive and insulin negative cells. Data are presented as mean ± SEM, and statistical significance was determined by a two-tailed T-test or one-way ANOVA with Tukey’s posthoc test.

[0060] FIGS.24A-24F show in vivo delivery and uptake of cargo by eGLP-LNPs to mouse pancreas. Fig.24A shows organ fluorescence imaging after intraperitoneal (IP) injection of various LNP conditions in 8-week-old C57BL / 6J male mice. Fluorescence radiant efficiency color scale measured in [p / sec / cm2 / sr] / [uW / cm2] and ranging from a minimum of 1.28e8 to a maximum of 7.09e8. Fig.24B shows average normalized expression measured by fluorescence quantification and calculated by using organ average radiant efficiency / average of PBS average radiant efficiency value of the pancreas. Fig. 24C shows average normalized expression measured by fluorescence quantification of the pancreas to liver average normalized fluorescent ratio. Fig.24D shows organ fluorescence imaging after IP injection of 8-week-old C57BL / 6J mice first injected with high dose streptozotocin (STZ, 180 mg / kg) and then various LNP conditions. Fluorescence radiant efficiency color scale measured as in Fig.24A. Fig. 24E shows the average normalized expression by fluorescence quantification of the pancreas in high-dose STZ-treated mice. Fig. 24F shows high dose STZ and LNP treated quantification of the pancreas to liver average normalized fluorescent ratio. Data are presented as mean ±SEM, and statistical significance was determined by a one-way ANOVA with Dunnett’s posthoc test.

[0061] FIGS.25A-25I shows pancreas-specific delivery of cargo with eGLP-lipid nanoparticles in NOD mice. Fig. 25A shows immunofluorescence imaging of islets from 6–12- week-old female NOD mice for glucagon-like peptide-1 receptor (GLP-1R, medium grey), insulin (INS, dark grey), and nuclei (DAPI, light grey). Scale bar = 100 nm. Fig. 25B shows quantification of percent GLP-1R positive cells within the islet. Values were calculated by taking GLP-1R-positive cells / DAPI-positive cells within the islet region of interest defined by insulin staining. Each dot represents individual islets separated by weeks of age. Figures 25C-25F show quantification of percent GLP-1R positive cells within the islet separated by severity of insulitis:MBHB Ref. No.24-0108-WO none (medium grey), moderate (light grey), severe (dark grey). Each dot represents individual islets. The dashed line represents the best-fit curve calculated by simple linear regression (R2value). Fig. 25G shows organ fluorescence imaging after IP injection of 6-week-old NOD mice with various LNP conditions. Fluorescence radiant efficiency color scale measured as in Fig. 24A. Fig.25H shows average normalized expression by fluorescence quantification of the pancreas. Fig.25I shows quantification of the pancreas to liver average normalized fluorescent ratio. Data are presented as mean ± SEM, and statistical significance was determined by a one- way ANOVA with Dunnett’s posthoc test.

[0062] FIGS.26A-26I show LNP-mediated -cell targeted delivery of a therapeutic mRNA. Fig.26A shows percent of MIN6 cells positive for programmed death-ligand 1 (PD-L1) after LNP delivery of PD-L1 mRNA encoded by CD274. Fig. 26B shows a schematic of an experimental timeline whereby 6-week-old female NOD mice were treated with various LNP conditions twice weekly for two weeks by IP injection. Fig. 26C shows diabetes incidence under the various LNP conditions. N=10 mice per group (Mantel-Cox). Fig.26D shows immunofluorescence imaging of islets from NOD mice for PD-L1 (medium grey), insulin (grey), and nuclei (DAPI, light grey). Scale bar=100 nm. Fig. 26E shows quantification of PD-L1 and insulin double-positive cells. Values were calculated by taking double positive-PD-L1 and insulin cells / total insulin-positive cells. Each dot represents measured individual islets. Fig.26F shows quantification of PD-L1 fluorescence intensity within the acinar compartment. Values calculated by PD-L1 mean fluorescence intensity / non-insulin-positive area. Each dot represents measured individual islets. Fig.26G shows immunohistochemistry imaging of the pancreas for insulin (dark grey) and nuclei (hematoxylin, medium grey). Fig. 26H shows -cell mass (mg). Each dot represents independent mice. Fig.26I shows insulitis scores. Each dot represents measured individual islets. Data are presented as mean ± SEM, and statistical significance was determined by a one-way ANOVA with Dunnett’s posthoc test.

[0063] FIGS.27A-27J show evaluation of LNP targeting specificity. Fig.27A shows organ fluorescence imaging quantification after IP injection of various LNP conditions in 8-week-old C57BL / 6J mice. Average normalized expression calculated by using organ average radiant efficiency / average of PBS average radiant efficiency value. The dots for each bar for each organ represent: PBS, empty, naked mRNA, LNP, and GLP1-LNP from left to right, respectively. Fig.27B shows non-fasted glucose measurements of C57BL / 6J mice treated with saline or high-MBHB Ref. No.24-0108-WO dose STZ over 72 hours. Fig. 27C shows body weight measurements of C57BL / 6J mice treated with saline or high-dose STZ over 72 hours. Fig.27D shows organ fluorescence imaging quantification after IP injection of high dose STZ and various LNP conditions in 8-week-old C57BL / 6J mice. Average normalized expression calculated by using organ average radiant efficiency / average of PBS average radiant efficiency value. The dots for each bar for each organ represent: PBS, naked mRNA, non-targeted, and targeted from left to right, respectively. Fig. 27E shows an average Glp1r mRNA expression dot plot of single-cell RNA-sequencing data from mouse islet atlas dataset. Fig.27F shows average Glp1r mRNA expression dot plot of single-cell RNA-sequencing data from NOD islet atlas dataset. Fig.27G shows percent prevalence of insulitis in NOD mice (from bottom to top). Fig. 27H shows organ fluorescence imaging after IP injection of various LNP conditions in 8 / 9-week-old female NOD mice. Fluorescence radiant efficiency color scale measured in [p / sec / cm2 / sr] / [uW / cm2]. Fig.27I shows the average normalized fluorescence quantification of the pancreas in treated mice. Average normalized fluorescence was calculated by using organ average radiant efficiency / average of PBS average radiant efficiency value. Fig.27J shows organ fluorescence imaging quantification after IP injection of various LNP conditions in 6-week-old NOD mice. None of the data showed significant differences, further highlighting that specificity of targeting peptide is localized to pancreas (for IVIS data). Data are presented as mean ± SEM.

[0064] FIGS.28A-28B show evaluation of PD-L1 effects after LNP delivery. Fig. 28A shows percent of MIN-6 cells positive for programmed death-ligand 1 (PD-L1) after LNP delivery of PD-L1 mRNA encoded by CD274. Fig. 28B shows normalized counts of Nanostring data of various immune markers within insulitic regions of interest from NOD mice treated with various LNP conditions. Data are presented as mean ± SEM, and statistical significance was determined by a one-way ANOVA with Dunnett’s posthoc test. DETAILED DESCRIPTION

[0065] Provided herein are compositions and methods for treating diabetes, including, for example, Type 1 diabetes (T1D).

[0066] As used herein, “diabetes” refers to the broad class of disorders characterized by impaired insulin production and glucose tolerance. Diabetes includes type 1 and type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, and impaired glucoseMBHB Ref. No.24-0108-WO tolerance. Type I diabetes is also known as Insulin Dependent Diabetes Mellitus (IDDM). The terms are used interchangeably herein. Type 2 is also known as Non- Insulin-Dependent Diabetes Mellitus (NIDDM)). As used herein, the term “a patient in need thereof” refers to an subject that has been diagnosed with type 1 diabetes, type 2 diabetes, gestational diabetes, pre- diabetes, insulin resistance, metabolic syndrome, or impaired glucose tolerance, or one that is at risk of developing any of these disorders. Patients in need of treatment also include those that have suffered an injury, disease, or surgical procedure affecting the pancreas, or individuals otherwise impaired in their ability to make insulin.

[0067] As used herein, “prediabetes” is a health condition characterized by a blood sugar

[0068] The term “preventing a disorder” as used herein, is not intended as an absolute term. Instead, prevention, for example, of type 1 diabetes (T1D) or type 2 diabetes (T2D), refers to delay of onset, reduced frequency of symptoms, or reduced severity of symptoms associated with the disorder. Prevention therefore refers to a broad range of prophylactic measures that will be understood by those in the art. In some circumstances, the frequency and severity of symptoms is reduced to non- pathological levels, e.g., so that the individual does not need traditional insulin replacement therapy.

[0069] It is to be understood that the particular aspects of the specification are described herein are not limited to specific embodiments presented, and can vary. It also will be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting. Moreover, particular embodiments disclosed herein can be combined with other embodiments disclosed herein, as would be recognized by a skilled person, without limitation.

[0070] Throughout this specification, unless the context specifically indicates otherwise, the terms “comprise” and “include” and variations thereof (e.g., “comprises,” “comprising,”MBHB Ref. No.24-0108-WO “includes,” and “including”) will be understood to indicate the inclusion of a stated component, feature, element, or step or group of components, features, elements or steps but not the exclusion of any other component, feature, element, or step or group of components, features, elements, or steps. Any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms, while retaining their ordinary meanings.

[0071] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indictates otherwise.

[0072] In some embodiments, percentages disclosed herein can vary in amount by ±10, 20, or 30% from values disclosed and remain within the scope of the contemplated disclosure.

[0073] Unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values herein that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0074] As used herein, ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. For example, “about 5%” means “about 5%” and also “5%.” The term “about” can also refer to ± 10% of a given value or range of values. Therefore, about 5% also means 4.5% - 5.5%, for example.

[0075] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.”

[0076] As used herein, the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio or which have otherwise been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0077] As used herein, the terms “therapeutically effective amount” or “effective amount” refer to that amount of a therapeutic agent, such as a nanoparticle including an RNA molecule encoding PD-L1, which when administered to a subject, is sufficient to effect treatment (e.g.,MBHB Ref. No.24-0108-WO improve symptoms) for a disease or disorder described herein, such as, for example, Type 1 diabetes (T1D). The amount of a compound or therapeutic agent which constitutes a “therapeutically effective amount” or “effective amount” can vary depending on the compound or therapeutic agent, the disorder and its severity, and the age, weight, sex, and genetic background of the subject to be treated, but can be determined by one of ordinary skill in the art.

[0078] As used herein, the term “dysfunctional pancreatic cell” refers to a metabolic disorder characterized by progressive loss or dysfunction of pancreatic cells. Pancreatic cells include, but are not limited to, endocrine cells such as beta cells, alpha cells, and delta cells; and exocrine cells.

[0079] As used herein, the terms “treating” or “treatment” refer to the treatment of a disease or disorder described herein, in a subject, preferably a human, and includes inhibiting, relieving, ameliorating, or slowing progression of the disease or disorder or one or more symptoms of the disease or disorder.

[0080] As used herein, the term “functionalized peptide” or “targeting peptide” is an agonist of a -cell specific receptor, such as glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), orFXYD domain containing ion transport regulator 2 a (FXYD2 a) that targets an LNP to a -cell.For example, a “peptide-functionalized lipid-based nanoparticle” can refer to an LNP that has been modified by the addition of a peptide that targets that LNP to a -cell. In this sense, the peptide is a -cell targeting peptide.

[0081] As used herein, the term “mRNA cargo” or “RNA molecule” is an RNA molecule that can encode a therapeutic protein, such as an immune checkpoint protein. Examples of contemplated immune checkpoint proteins include programmed death ligand-1 (PD-L1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain-containing protein 3 (TIM3) and other T cell receptors. In other instances, an mRNA cargo can encode immune attenuating cytokines, such as IL-12 or IL-23 or -cell transcriptional factors, such as Paired Box 6 (PAX6) or V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAF-B).

[0082] As used herein, “nucleic acid” means a polynucleotide such as a single or double- stranded DNA or RNA molecule, including, for example, genomic DNA, cDNA, and mRNA. The term nucleic acid includes nucleic acid molecules of both natural and synthetic origin, asMBHB Ref. No.24-0108-WO well as molecules of linear, circular, or branched configuration representing either sense or antisense strands, or both, of a native nucleic acid molecule.

[0083] As used herein, “delaying” the onset of diabetes means to defer, hinder, slow, retard, stabilize, and / or postpone development of the disease. This delay can be of varying lengths of time, depending on the history of the disease and / or individual being treated. As is evident to one skilled in the art, a sufficient or significant delay can, in effect, encompass prevention, in that the individual does not develop the disease. A method that “delays” development of diabetes is a method that reduces probability of disease development in a given time frame and / or reduces the extent of the disease in a given time frame, when compared to not using the method. Such comparisons are typically based on clinical studies, using a statistically significant number of subjects.

[0084] As used herein, the term “subject” refers to a warm blooded animal such as a mammal, preferably a human, which is afflicted with, or has the potential to be afflicted with one or more diseases and disorders described herein.

[0085] As used herein, the term “pharmaceutical composition” refers to a composition that can include a lipid, a targeting peptide, and one or more therapeutic agents disclosed herein, such as an RNA molecule encoding PD-L1, and optionally, a pharmaceutically acceptable carrier, a solvent, an adjuvant, and / or a diluent, or any combination thereof.

[0086] In view of the present disclosure, the methods and compositions described herein can be configured by the person of ordinary skill in the art to meet the desired need. In general, the disclosed materials and methods provide improvements in treating diabetes as described herein.

[0087] A hallmark of T1D is insulitis, where immune cells infiltrate pancreatic islets and destroy the insulin-secreting cells. Insulitis is central in driving T1D progression. However, human studies indicate that insulitis is transient with relatively few islets displaying concurrent insulitis5,6,73. Moreover, in islets without insulitis, cells still appear to be dysfunctional73. As a result, therapies targeting only the immune response may not effectively treat T1D. New evidence suggests that cells are active participants, not innocent bystanders, during immune attack11. Under stress, cells can produce aberrant proteins that act as neoantigens to initiate or exacerbate autoimmunity9,8,10. The pathogenesis of T1D can be viewed as a “circuit” whose components feed forward to propagate signals from environment (macrophages,inflammation) cells (stress) dendritic cells (antigen presentation) adaptive immune cellsMBHB Ref. No.24-0108-WO(T / B cells) cells (death) (Fig. 1). Depending upon timing, intervening in the communicationbetween these cells at any point should “short the circuit” and reduce the risk or progression of T1D. However, most trials to date have focused on the adaptive immune component2-4.

[0088] Recent years have seen a renaissance in the understanding of the pathogenesis of T1D. The notion that T1D is typified by aggressive T cell-mediated insulitis and rapid loss of - cell mass has not proved to be uniformly true in humans5,6,66,67. This revelation contributes a new perspective on the pathogenesis of T1D— that intracellular signals arising from the cell stress responses can lead to the production of aberrant proteins that act as neoantigens to initiate or exacerbate autoimmunity (Fig.5)9-11. Thus, cells may mediate their own demise. Hints that cell interventions have benefits on their own have been suggested in preclinical studies (e.g. studies of senescence, inflammation and ER stress)17-19and in clinical studies (e.g. verapamil, imatinib)74,75. This understanding of the pathogenesis of T1D invites new opportunities to develop therapeutics that target the health and signaling responses of cells. In the future, combination immune cell- and cell-targeted therapeutics could be used to more effectively treat T1D. This present study focuses on developing precision nanomedicine technology to deliver biologic therapeutics directly to cells.

[0089] Recent research has identified novel targets and pathways that can promote the regeneration and survival of cells. However, it has been observed that many of these targets and pathways lack specificity to islet / cells and may raise safety concerns when delivered systemically, potentially affecting non-pancreatic cells negatively. Targeted drug delivery offers an opportunity to confine regenerative or survival therapies to islet / cells, thereby minimizing or avoiding adverse effects on tissues or cells outside the pancreas.

[0090] In this disclosure, a milestone-driven series of experiments was engaged with the goal to test the feasibility of peptide-functionalized lipid-based nanoparticles to target cells in vitro and in vivo (see Examples below). The goal focused on targeting technologies, rather than specific cargo. Nevertheless, this present disclosure uses delivery of nucleic acid cargo (mRNAs, RNA-interference, antisense oligonucleotides, and CRISPR) to specifically engage intracellular targets and monitor biological outputs. It was hypothesized that peptide- functionalized nanoparticles can be used to deliver RNA cargo specifically to cells to allow T1D disease modification. Both mouse and human systems were leveraged in this approach to ensure (1) that preclinical model systems were tested for specific cell targeting both in vitroMBHB Ref. No.24-0108-WO (isolated mouse and human islets) and in vivo (mice), and (2) that the cell targeting approach can prevent and / or reverse T1D in the NOD mouse model (Fig. 3 and Fig. 4).

[0091] To test the ability of peptide-functionalized nanoparticles to target cells in vitro, we focused on developing nanoparticles that are functionalized with known peptides that interact with glucagon-like peptide-1 (GLP-1R)21,22(Fig.2) and identifying new peptides that interact with ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), FXYD domain containingion transport regulator 2 a (FXYD2 a), and GLP-1R by phage display. GLP-1R is the mosthighly enriched receptor that is specific to pancreatic cells. Hence, GLP-1R was the first amongst the top candidates to be investigated. The functionalized nanoparticles were complexed with fluorescent-encoding mRNA to demonstrate delivery of this cargo to mouse and human cells (islets and cell lines) in vitro (Fig.3, Fig.8, and Fig.9).

[0092] Furthermore, the next goal was to test the feasibility of peptide-functionalized nanoparticles to direct cell-specific cargo delivery in vivo (Fig.3, Fig.4, and Fig.16). This goal was to advance the most promising functionalized nanoparticles from the in vitro experiments into systems in vivo: normal mice, “humanized” mice (immunodeficient mice transplanted with human islets), and NOD mice. In NOD mice, PD-L1-encoding mRNA was delivered to cells to test for prevention / reversal of T1D. Subsequent analyses included pathologic verification of -cell targeting (by immunostaining of tissue), intravital imaging of endogenous and transplanted islets in live mice, and assessment of diabetes prevention / reversal.

[0093] Disclosed herein is a targeted approach through delivery of therapeutic agents, such as bioactive molecules, to key pancreatic cell types, particularly cells to treat diabetes in a subject. The engineered targeted nanoparticles successfully deliver therapeutic agents, such as bioactive molecules, to key pancreatic cell types, particularly cells via a targeting peptide against glucagon-like peptide-1 receptor (GLP-1R). Examples (Examples 3, 4, 6, 7) using non- diabetic models demonstrated in vitro -cell line targeting and in vivo delivery of LNPs to the pancreas with successful protein production from mRNA cargo (Examples 8-10). This targeting also successfully delivered cargo to human EndoC- H1 cell line.

[0094] The significance of the present disclosure includes at least two aspects. First, it provides novel nanomedicine approaches to treat dysfunctional pancreatic cells and diabetes with unmet medical need. Second, it integrates targeted nanomedicine and RNA therapeutics to create a new avenue for the treatment of diabetes, including, for example, Type 1 diabetes (T1D). ThisMBHB Ref. No.24-0108-WO disclosure further provides, in part, a peptide-targeted complex using lipids to deliver therapeutic mRNA to cells, delivering Cd274 to -cells, promoting -cell survival, restoring functional - cells, reduce insulitis, and delaying disease progression.

[0095] Compositions

[0096] In some embodiments, pharmaceutical compositions contemplated herein include a therapeutically effective amount of targeted nanoparticles (e.g., LNPs) including a functionalized peptide, such as glucagon-like peptide-1 (GLP-1), and an RNA molecule encoding an immune checkpoint protein, e.g., programmed death ligand-1 (PD-L1). Such compositions may further include an appropriate pharmaceutically acceptable carrier, solvent, adjuvant, diluent, or any combination thereof. The exact nature of the carrier, solvent, adjuvant, or diluent will depend upon the desired use (e.g., route of administration) for the composition, and may range from being suitable or acceptable for veterinary uses to being suitable or acceptable for human use.

[0097] In some embodiments, pharmaceutical compositions contemplated herein include one or more types of LNPs that may vary by composition, such as lipid compostion, and that carry a functionalized peptide, such as glucagon-like peptide-1 (GLP-1); and an RNA molecule, such as, for example, an RNA molecule encoding PD-L1, for example, inside the nanoparticle, attached to an external surface of the nanoparticle, or both. In some embodiments, the LNPs include one or more targeting moeities attached thereto to enable targeted delivery of the LNPs to a desired location. For example, the targeting moeity (e.g., a protein or peptide) can target the LNPs to - cells in the pancreas.

[0098] Any therapeutic mRNA is contemplated herein. For example, contemplated mRNAs include programmed death ligand-1 (PD-L1), cytotoxic T-lymphocyte associated protein 4 (CTLA-4), lymphocyte activation gene 3 (LAG-3), T cell immunoglobulin and mucin domain- containing protein 3 (TIM3) and other T cell receptors, or immune attenuating cytokines, such as IL-12 or IL-23 or -cell transcriptional factors, such as Paired Box 6 (PAX6) or V-maf musculoaponeurotic fibrosarcoma oncogene homolog B (MAF-B).

[0099] Any functionalized / targeting peptide is contemplated herein. For example, contemplated functionalized / targeting peptides include an agonist of a -cell specific receptor, such as glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), or an FXYD domain containing iontransport regulator 2 a (FXYD2 a).MBHB Ref. No.24-0108-WO [000100] Such compositions optionally include secondary therapeutic agents (possibly also carried on or in contemplated LNPs). [000101] In some embodiments, LNPs carring an RNA molecule encoding PD-L1, of the present disclosure can be administered through a variety of routes and in various compositions. For example, pharmaceutical compositions containing LNPs harboring the RNA molecule (e.g., an mRNA) encoding PD-L1 can be formulated for oral, intravenous, topical, ocular, buccal, systemic, nasal, injection, transdermal, rectal, or vaginal administration, or formulated in a form suitable for administration by inhalation or insufflation. In some embodiments of the present disclosure, administration is oral or intravenous. [000102] A variety of dosage schedules is contemplated by the present disclosure. For example, a subject can be dosed monthly, every other week, weekly, daily, or multiple times per day. Dosage amounts and dosing frequency can vary based on the dosage form and / or route of administration, and the age, weight, sex, and / or severity of the subject’s disease. In some embodiments of the present disclosure, pharmaceutical compositions of LNPs carring one or more therapeutic agents, are administered orally, and the subject is dosed on a daily basis. [000103] The therapeutic agents (also referred to as “compounds” herein) described herein (e.g., lipid LNP, functionalized peptide, mRNA), or compositions thereof, will generally be used in an amount effective to achieve the intended result, for example, in an amount effective to provide a therapeutic benefit to subject having the particular disease being treated. As used herein, therapeutic benefit refers to the eradication or amelioration of the underlying disease being treated and / or eradication or amelioration of one or more of the symptoms associated with the underlying disease such that a subject being treated with the therapeutic agent reports an improvement in feeling or condition, notwithstanding that the subject may still be afflicted with the underlying disease. [000104] Determination of an effective dosage of compound(s) for a particular disease and / or mode of administration is well known. Effective dosages can be estimated initially from in vitro activity and metabolism assays. For example, an initial dosage of compound for use in a subject can be formulated to achieve a circulating blood or serum concentration of the therapeutic compound that is at or above an IC50 of the particular compound as measured in an in vitro assay. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of the particular compound via a given route of administration is wellMBHB Ref. No.24-0108-WO within the capabilities of a skilled artisan. Initial dosages of compound can also be estimated from in vivo data, such as from an appropriate animal model. [000105] Dosage amounts of an RNA molecule encoding PD-L1 can be in the range of from about 0.0001 mg / kg / day, about 0.001 mg / kg / day, or about 0.01 mg / kg / day, or about 0.1 mg / kg / day, or about 1.0 mg / kg / day, or about 10 mg / kg / day to about 100 mg / kg / day, but may be higher or lower, depending upon, among other factors, the activity or expression level of the active compound, the bioavailability of the compound, its metabolism kinetics and other pharmacokinetic properties, the mode of administration and various other factors, including particular condition being treated, the severity of existing or anticipated physiological dysfunction, the genetic profile, age, health, sex, diet, and / or weight of the subject. Dosage amounts and dosing intervals can be adjusted individually to maintain a desired therapeutic effect over time. For example, the compounds may be administered once, or once per week, several times per week (e.g., every other day), once per day or multiple times per day, depending upon, among other things, the mode of administration, the specific indication being treated and the judgment of the prescribing physician. In cases of local administration or selective uptake, such as local topical administration, the effective local concentration of compound(s) and / or active metabolite compound(s) may not be related to plasma concentration. Skilled artisans will be able to optimize effective dosages without undue experimentation. [000106] For example, a dosage contemplated herein can include a single volume of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, or 3.0 mL of a pharmaceutical composition having a concentration of mRNA encoding PD-L1 at about 0.00001, 0001, 0.001, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 10, 15, 20, 50, 100, 200, 500, or 1000 mM in a pharmaceutically acceptable carrier. [000107] Nanoparticles [000108] The present disclosure contemplates use of lipid nanoparticles (LNPs, also referred to as nanoparticles herein) to deliver therapeutic agents. LNPs are innovative delivery systems that encapsulate nucleic acids to facilitate targeted gene therapy, offering a non-viral, biocompatible alternative for treating various diseases. LNPs commonly comprises 4 key elements. In addition to an ionizable or cationic lipid to encapsulate nucleic acids, additional components such as phospholipids, cholesterol, and polyethylene glycol PEG-functionalized lipid (PEG-Lipid) can help determine LNP performance. Phospholipid DOPE, which serves as the key component ofMBHB Ref. No.24-0108-WO biological membranes and organelles, aids in nucleic acid encapsulation and tends to form a conical shape to disrupt endosomal membrane to facilitate endosomal escape. Cholesterol enhances LNP stability by modulating membrane integrity and rigidity. PEG-Lipid contributes to nanoparticle stability and blood circulation. PEG-Lipid allows chemical modification to endow the LNPs with targeted delivery of nucleic acids to specific tissues or cells of interest. Inclusion of these components at an optimized ratio promotes LNP delivery efficiency, stability, and biodistribution. [000109] The contemplated LNPs can comprise 1,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000 (DSPE-PEG). DSPE domains promote nanoparticles formation and facilitate nanoparticle stability. PEG domains prevent macrophase separation, stabilizing the LNPs. The domains further protect the nanoparticles from recognition by the reticuloendothelial system in the body. The PEG domain can be comprised of PEG having an average molecular weight of about 1,000 to about 100,000 Daltons (Da). [000110] Contemplated nanoparticles for use herein include, for example, LNPs that can effectively incorporate negatively-charged nucleotides and functionally display tissue-targeting peptides on the surface. These self-assembled nano-scale carriers (~100 nm in diameter) are formed through self-assembly. In one embodiment, LNPs include a lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14), cholesterol, 1, 2-Distearoyl-sn-glycero-3-phosphoethanolamine- Poly(ethylene glycol) (DSPE-PEG), dioleoylphospha-tidylethanolamine (DOPE), and PD-L1 mRNA and a targeting peptide that is conjugated to the PEG-lipid to functionalize the nanoparticles to bind specific cell membrane molecules. The contemplated LNPs can comprise a lipid, a GLP-1 targeting molecule, and an RNA molecule encoding PD-L1. Negatively-charged nucleotides are encapsulated in the cores of the nanoparticles. This approach offers multiple advantages, including: (i) the nano-scale of size significantly increases the surface area : volume ratio that can enhance specific targeting, and (ii) the self-assembling feature of the LNP eliminates the use of chemical cross-linking agents, thereby reducing possible toxicities. [000111] Additional LNPs are contemplated for use herein, such as those disclosed in U.S.Patent No. 9,505,867, Vieregg et al. (J. Am. Chem. Soc. 2018, 140, 1632 1638), Lueckheide etal. (Nano Lett. 2018, 18, 7111 7117), and Marras et al. (Polymers 2019, 11, 83), each of whichis incorporated by reference. [000112] Targeting MoleculesMBHB Ref. No.24-0108-WO [000113] The present disclosure contemplates use of targeting molecules (or targeting moieties / peptides) with the nanoparticles disclosed herein for targeted delivery of therapeutic compositions, such as an RNA molecule encoding PD-L1 or other therapeutic protein or peptide, or for incorporation into pharmaceutical compositions as described herein. Targeting molecules can be GLP-1-targeting molecules. Targeting molecules can include peptides such as AibEGTFTSDVSSYLEEQAA-KEFIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 1), which is an agonist of the GLP-receptor. [000114] In one embodiment, a contemplated targeted nanoparticle contains an RNA molecule encoding PD-L1 (1×10-13-1×10-8μM) having the nucleotide sequence of SEQ ID NO: 2 (NM_014143.4). [000115] In some embodiments, contemplated nanoparticles containing an RNA molecule encoding PD-L1 exhibit a polydispersity of about 0.1 to about 0.3. [000116] In some embodiments, contemplated nanoparticles containing an RNA molecule encoding PD-L1 contained with the core exhibit a spherical shape and have a diameter (in nanometers, nm) of about 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 nm. [000117] The nanoparticle delivery system of the present disclosure possesses multiple advantages compared with other nanoparticle-based platforms. The first advantage is higher stability: the therapeutically active components (e.g., an RNA molecule encoding PD-L1, or other nucleic acid based therapeutics) can be encapsulated in the inner core of the LNP and can therefore be protected by the outer layer of biocompatible polymers. Through this approach, first, the degradation of nucleotides of the RNA molecule encoding PD-L1 by serum nucleases is prevented; second, the LNPs are capable of escaping renal clearance; and third, immunogenic responses are avoided. The second advantage is higher safety: cell-targeting peptides (e.g., those targeting to GLP-1 are covalently conjugated on the periphery of the LNP, which significantly reduces cytotoxicity and increases circulation time by circumventing nonspecific interaction with serum components. The third advantage is higher specificity: with the defined chemical structures of the targeting peptides, the LNPs are able to bind specific receptors and penetrate targeted cells. An additional advantage is higher scalability. This approach does not require chemical modifications on nucleotides for conjugation, nor does it need to engineer hard-to-MBHB Ref. No.24-0108-WO reproduce lipid nanoparticles. The synthesis of the core components in the LNPs is highly automated. In addition, the targeting peptides are easily changeable to target different receptors. [000118] Further, as shown herein, the use of targeted nanoparticles permits use of a lower amount of a therapeutic agent for the treatment of a diabtetes due to the specific targeting of the therapeutic agent to the pancreas. In this way, use of targeted nanoparticles can significantly lower the dosage of a therapeutic agent required to treat diabetes, which can significantly reduce costs associated with the treatment. For example, a therapeutically effective amount of a therapeutic agent to be delivered by a targeted nanoparticle can be at least about 10, 20, 30, 40, or 50% lower than the therapeutically effective amount of the naked (non-targeted) therapeutic agent. [000119] Lipid nanoparticles are colloidal dispersions that are composed of one or more lipid- bilayers that surround an aqueous core. The ability of LNPs to encapsulate lipophilic and hydrophilic drugs have allowed these vesicles to be useful drug delivery systems. Important physicochemical properties of LNPs such as the hydrodynamic diameter or particle size, surface charge (typically measured as zeta-potential), lipid-packing, bilayer lamellarity, encapsulation efficiency, drug encapsulation, molecular loading and external modifications (such as polymer coatings and targeting moiety incorporation) are necessary to accurately control and measure to properly manufacture a pharmaceutical drug product. Lipid nanoparticles can be formed to have a hydrodynamic diameter (in nanometers [d.nm]) ranging from approximately 30 d.nm to over 1,000 d.nm. For lipid nanoparticles that are less than 1,000 d.nm, these particles exhibit Brownian motion and remain as a colloidal dispersion since the thermal motion of the particles overcome gravitational forces that would otherwise increase the likelihood of sedimentation. [000120] Methods [000121] In some embodiments, methods of treating and / or preventing diabetes in a subject in need thereof include administering to the subject a therapeutically effective amount of PD-L1 or other nucleic acid based therapeutic. Treatable and / or preventable diabetes can include Type 1 diabetes (T1D). [000122] In some embodiments, therapeutic methods contemplated herein can also treat and / or prevent complications associated with pancreatic disorders by administering to the subject a therapeutically effective amount of PD-L1 or another therapeutic. For example, dysfunction ofMBHB Ref. No.24-0108-WO pancreatic cells plays a pivotal role in the development of diabetes. For example, Type 1 diabetes (T1D) is an autoimmune disease in which the body’s immune system mistakenly targets and destroys insulin-producing cells in the pancreas. In Type 2 diabetes (T2D), the pancreas produces less insulin than needed, and the body becomes resistant to its effects. Without wishing to be bound by theory, it is believed that targeting -cells associated with pancreatic disorders, such as diabetes, for treatment and / or prevention of complications associated with pancreatic disorders (e.g., by reducing or preventing pancreatic inflammation by increasing PD-L1 expression in the pancreas) can promote cell survival and restore functional cells, reduce insulitis, and delay disease progression. Each of these benefits can have a transformative impact on the treatment of diabetes. [000123] The present disclosure contemplates a variety of methods of administering the therapeutic agents, targeting molecules, and lipids disclosed herein, including local, oral, nasal, rectal, intravaginal, topical, subcutaneous, intradermal, intramuscular (IM), intravenous (IV), intrathecal (IT), intracerebral, epidural, or intracranial administration. Local, in situ administration of these compositions is contemplated. [000124] The present disclosure contemplates methods that result in a variety of indications of improvement for pancreatic disorders in the patient. [000125] The present disclosure contemplates methods that result in a variety of indications of improvement for pancreatic disorders, such as diabetes, in the patient. [000126] The present disclosure contemplates methods that result in a variety of indications of improvement for Type 1 diabetes (T1D) in the patient. [000127] The present disclosure contemplates use of the disclosed methods in conjunction with other treatments for diabetes in the patient. [000128] The present disclosure contemplates methods that result in a variety of indications of improvement or delay an onset of diabetes in a patient. EXAMPLES [000129] The Examples that follow are illustrative of specific embodiments of the disclosure, and various uses thereof. They are set forth for explanatory purposes only and should not be construed as limiting the scope of the disclosure in any way.MBHB Ref. No.24-0108-WO [000130] A driving force in type 1 diabetes (T1D) progression is insulitis, wherein immune cells infiltrate pancreatic islets and destroy the insulin-secreting -cells1. While suppressing this immune response has been the focus of most clinical trials2–4, this strategy has not significantly improved diabetes outcomes. Recent studies suggest that the autoimmune response is not the only contributor to T1D pathogenesis. Human studies show that relatively few islets exhibit insulitis5,6; meanwhile, most islets (even those lacking insulitis) exhibit -cell dysfunction2. These dysfunctional -cells can produce aberrant proteins that act as neoantigens to initiate and / or exacerbate autoimmunity8-10. Thus, -cells appear to be active participants in disease progression, not just innocent bystanders during autoimmune attack11. With knowledge of these critical -cell advancements, there is increasing interest in developing therapeutics to improve - cell health. Some possibilities include drugs that enhance -cell function like imatinib and verapamil13,15or other drugs that target -cell stress pathways of senescence18, the unfolded protein response17,19, and proinflammatory signaling19. However, a major limitation of these drugs is that their targets are not specific to -cells, which could result in significant off-target effects with systemic drug delivery. Currently, there is a critical gap in technology that prevents the ability to therapeutically target the -cell and, ultimately, translate this innovative approach into a potential clinical treatment for diabetes. [000131] One exciting tool that may be used to achieve -cell-targeted therapies is lipid nanoparticle (LNP) technology. LNPs can safely and effectively deliver various therapeutic cargo (e.g., mRNA, bioactive peptides, and CRISPR products25) and can be customized to precisely target any specific cell type of interest27. LNP technology has gained momentum due to its success in COVID-19 vaccines and cancer therapy30,97,98. These advancements stand at the forefront of therapeutic innovation that promises a new era of precision medicine for multiple diseases. Therefore, now is the time to harness this technology to target -cells in T1D. [000132] One method in which LNP technology could be used to revolutionize diabetes treatment is by increasing -cell specific expression of programmed death ligand-1 (PD-L1). Normally expressed on the cell surface of immune cells, PD-L1 binds to its receptor programmed cell death protein-1 (PD-1) leading to an inhibitory signal that suppresses the immune response46. It has been shown that cancer cells exploit this immune checkpoint system by expressing PD-L1 on the cell surface to evade immune detection99. During T1D progression, PD-L1 expression has been shown to increase in a subset of -cells that evade T-cell mediatedMBHB Ref. No.24-0108-WO insulitis and cell death19,47. When PD-L1 is expressed on human islet-like organoids, PD-L1 overexpression restores glucose homeostasis in immune competent diabetic mice63. Therefore, increasing PD-L1 expression in -cells with cell-targeted LNPs could enable safe and effective preservation of -cell function and delay T1D onset. [000133] The present examples are directed to an innovative therapeutic tool that leverages LNP technology to target -cells and to deliver a therapeutic cargo (e.g., a nucleic acid encoding PD-L1 or other therapeutic protein or peptide). To successfully target the LNPs to -cells, the LNPs are conjugated with a glucagon-like peptide-1 (GLP-1). GLP-1 has previously been used to successfully deliver antisense oligonucleotides to -cells22, making it a promising ligand to direct drug delivery from LNPs. For therapeutic cargo, a cluster of differentiation 274 (Cd274) mRNA, which encodes the aforementioned PD-L1 immune checkpoint protein is incorporated (Fig.11). Together, these individual components (LNP technology, GLP-1 functionalization, and mRNA cargo encoding PD-L1) represent an innovative treatment strategy tested in a mouse model of T1D. Lastly, this work establishes the feasibility of targeting -cells with LNP technology and implementing such technologies for treatment of T1D in humans. [000134] Materials and Methods [000135] Lipid Nanoparticle Formulation and Synthesis: Lipids containing G0-C14 (seebelow), 18:1 ( 9-C1s) PE (DOPE; Avanti Polar Lipids), cholesterol (Sigma), and DSPE-PEG2K(Mw=2000; Nanosoft Polymers) were dissolved in ethanol and mixed together at a molar ratio of 5:15:25:1 to assemble the unconjugated LNP as described previously104. For the eGLP1 targeted LNP (eGLP-LNP / conjugated LNP) was formulated by an identical method except that 30% of the DSPE-PEG2K was replaced with DSPE-PEG-eGLP-1 (see below). The lipids were mixed with nuclease-free DI water containing mRNA (Cy5-EGFP mRNA; APExBIO, R1011) and Cd274; (GenScript, SEQ ID NO: 2 and SEQ ID NO: 4) at a volume ratio of 3:1 (aqueous: ethanol=3:1, v / v). The ratio of nitrogen present on cationic lipid (N:P) was 8.5. The formed LNPs were then dialyzed with sterilized PBS for 2 h to remove ethanol by a Slide-A-Lyzer dialysis cassette with MWCO=20K (ThermoScientific, 66003). To generate the DSPE-PEG- eGLP1 LNPs, 100 mg DSPE-PEG2K-MAL (Mw=2000; Nanosoft Polymers) was dissolved in 3 mL DI water containing 50% EtOH. Then, 142 mg eGLP-1 peptide (eGLP-1 peptide sequence: H(Aib)EGTFTSDVSSYLEEQAAKEFIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 1), GenScript)34,105containing 20 mg Tris(2-carboxyethyl) phosphine hydrochloride (TCEP; Sigma)MBHB Ref. No.24-0108-WO and 3 mM EDTA•2Na (Sigma) was added to the above solution and the reaction continued for 24 hours. The products were intensively dialyzed (MWCO=1000) with DI water and lyophilized to obtain the DSPE-PEG-eGLP-1 LNPs. All the reactions were conducted under N2 atmosphere. The cationic lipid G0-C14 was synthesized through the reaction of PAMAM G0 dendrimer (Sigma-Aldrich) and 1,2-Epoxytetradecane (Sigma-Aldrich) according to previously published methods95,106. Briefly, 1,2-Epoxytetradecane was added into PAMAM G0 dendrimer as a mass ratio of 6:1. The reaction was carried out for 2 days under vigorous stirring. The product was future separated on silica with gradient elution from CH2Cl2to CH2Cl2: MeOH:NH4OH with 75:22:3 (v / v). [000136] Lipid nanoparticle Characterization: Transmission electron microscope (TEM) images were obtained using Spirit LaB6 Electron Microscope (FEI Company) with a voltage of 120 kV located in the Advanced Electron Microscopy Facility at the University of Chicago. NMR spectrum was recorded by a 400 MHz NMR instrument instrument located in the NMR facility at the University of Chicago. The hydrodynamic size and zeta potential was measured bya Wyatt Möbiu instrument (Wyatt Technology) located in the Soft Matter CharacterizationFacility at the University of Chicago. Encapsulation efficiency was quantified by RiboGreen assay kit (Invitrogen, R11490). [000137] GLP-1R Total Internalization Assay: Measurement of GLP-1R total internalization utilized the PathHunter® eXpress GLP1R U2OS Total GPCR Internalization Assay (Eurofins DiscoverX Corp; 93-1075E3). Exendin-4 was used as a control agonist (100 nM, Eurofins DiscoverX Corp, 92-1115). Normal protocols were followed using manufacturer provided procedure. In brief, cells were plated into 96-well format and incubated for 48 hours at 37°C. Then various conditions were added to the media for 3 hours, and the experiment was stopped by using the included PathHunter Detection Kit. Chemiluminescence was measured using Spark Microplate Reader (Tecan Trading AG). [000138] Animals and procedures: Mouse experiments were performed under specific pathogen-free conditions and maintained in 12 h:12 h light:dark cycle with free access to food and water as per protocols approved by the University of Chicago Institutional Animal Care and Use committee. CD1 mice were purchased from Charles River (Charles River #022), and NOD / ShiLtJ (NOD; Jackson Labs #1976) mice, and C57BL / 6J (B6; Jackson Labs #000664)MBHB Ref. No.24-0108-WO mice were purchased and allowed a minimum of 72 h recovery prior to experimentation. At the end of each study, mice were euthanized, and tissues and blood were collected. [000139] To isolate mouse islets, collagenase was injected into the pancreatic bile duct to inflate the pancreas prior to removal as previously described107. Briefly, a Histopaque-HBSS gradient was applied to the dissociated pancreas, followed by centrifugation at 900 x g for 18 min. The mouse islets were then removed from the center of the gradient and cultured in RPMI medium. Islets were allowed to recover overnight before in vitro experimentation. [000140] Diabetic Model Treatments and Organ Biodistribution: 8–10-week-old male C57BL / 6J mice were injected with either water or a single dose of 180 mg / kg of streptozotocin (STZ) by IP injection. Random-fed glucose levels were measured by tail snip using a glucometer (AlphaTrak), and mice were followed for 3 days post STZ injection. Upon two consecutive days of blood glucose values greater than 250 mg / dL, mice were deemed diabetic and LNPs (1 mg / kg) were injected. At the end of the study, mice were euthanized, and tissue and blood samples were collected. [000141] For therapeutic experiments and diabetes incidence related to CD274 delivery, 6- week-old female NOD mice were injected twice weekly for two consecutive weeks with PBS, eGLP-LNPs + no cargo, unconjugated LNPs + CD274, or eGLP-LNPs + CD274 at a dose of 1 mg / kg by IP injection. CD274 was purchased from GenScript (SEQ ID NO: 2 and SEQ ID NO: 4). At the end of the treatment period, mice were euthanized, and tissue was collected. [000142] For organ biodistribution analysis, adult C57BL / 6J or NOD mice were IP injected with relevant experimental conditions (PBS, naked mRNA cargo (1 mg / kg), LNP + mRNA, or eGLP-1 LNP + mRNA). For fluorescent delivery, Cy5-EGFP mRNA was injected (APExBIO, R1011). After 18 hrs of experimental condition exposure, mice were euthanized and the heart, lung, liver, spleen, kidney, and pancreas were harvested for the lipid nanoparticle biodistribution analyses. Fluorescence imaging of organs was conducted using an in vivo imaging system (IVIS 200, PerkinElmer). [000143] Biodistribution of LNPs for in vivo mouse models: Adult C57BL / 6J, CD1, or NOD mice were intraperitoneally injected with relevant experimental conditions (PBS, naked mRNA cargo (0.8 mg / kg bogy weight), non-targeted LNPs + mRNA, or targeted LNPs + mRNA). After 18 hrs of experimental condition exposure, mice were euthanized and the heart, lung, liver, spleen, kidney, and pancreas were harvested for the liposomal nanoparticles biodistributionMBHB Ref. No.24-0108-WO analyses. Fluorescence imaging of organs was conducted using an in vivo imaging system (IVIS 200, PerkinElmer). [000144] Human islets: De-identified non-diabetic male and female human donor islets were obtained from the Integrated Islet Distribution Program (IIDP) and the University of Alberta Diabetes Institute Islet core. The use of de-identified human samples was approved by the Institutional Review Board at the University of Chicago and considered exempt from human subjects research. [000145] Cell culture and treatment: MIN6108,109mouse cells were cultured in high glucose (25 mM) Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 15% fetal bovineserum, 1% penicillin / streptomycin cocktail (P / S), and 1% L-Glutamine. Human EndoC- H1cells110(69) were cultured in low glucose DMEM (5.5 mM) supplemented with 2% BSA, 50 M -mercaptoethanol, 10 mM 20 nicotinamide, 5.5 g / ml transferrin, 6.7 ng / mL sodium selenite, and 1% P / S, in plates pre-coated with matrigel-fibronectin. HEK-293 cells were cultured in high glucose DMEM supplemented with 10% FBS, 1% P / S, and 1% L-Glutamine. Mouse islets were cultured in RPMI medium supplemented with 10% FBS and 1% P / S. [000146] To test LNPs in vitro, 300 ng mRNA (either Cy5-eGFP or Cd274) was used andtreated MIN6, or EndoC- H1 under multiple timepoints listed in text (hrs to overnight). ForExendin-4 (Ex-4; Sigma, E7144)) studies, cells were pretreated with vehicle (H2O), 1 nM Ex-4, or 10 nM Ex-4 for 1 h followed by co-treatment with LNP conditions overnight. [000147] Flow Cytometry: Dissociated cells were placed in Stain Buffer (BD Pharmigen, 554657) and processed with BD Perm / Wash Buffer (BD Pharmigen, 554723) for intracellular staining according to manufacturer’s protocol. Cells were incubated with primary antibodies for Insulin (1:100, rb, Cell Signaling, 4590S), Glucagon (1:100, ms, Abcam, ab10988), or PD-L1 (R&D Systems, FAB1562R; 1:100). Secondary antibodies were conjugated to corresponding primary antibody organism (ThermoFisher; 1:100). After washing, analysis of cells was performed on Beckman Coulter CytoFLEX S and data was analyzed using FlowJo (v.10.1.1). [000148] Protein isolation and immunoblotting: Protein was isolated and western blots were performed using standard procedures. Briefly, whole-cell extracts of cells were prepared in a lysis and extraction buffer (ThermoFisher) supplemented with HALT protease inhibitor cocktail (ThermoFisher) and protein extract was resolved by electrophoresis on a precast 4-20% tris- glycine polyacrylamide gels (Bio-Rad), transferred to polyvinylidene difluoride membrane, andMBHB Ref. No.24-0108-WO membranes were blocked with Intercept® (TBS) blocking buffer (Li-Cor Biosciences) for 1-2 h. The blots were probed with the following primary antibodies and 0.2% Tween20 with overnightincubation at 4°C: anti-PD-L1(Cell Signaling; 13684; 1:1000), and anti- -actin (Cell Signaling;3700s; 1:1000). Anti-rabbit or anti0 mouse (Li-Cor BioSciences; 1:10000) secondary antibodies were used for visualization and quantification. Immunoblots were visualized using the Li-Cor Odyssey system (Li-Cor Biosciences) and quantitated using Odyssey Imaging software (Li-Cor Biosciences). [000149] Immunofluorescence staining and Quantification: Pancreata were fixed in 4% paraformaldehyde, paraffin embedded, and sectioned at 5 mm. For immunofluorescence staining, pancreata were stained for using the following antibodies: anti-PD-L1 (rb, Abcam; ab213480; 1:200), anti-insulin (gp, Dako, IR002; 1:5), anti-GLP-1R (gt, Origene, TA326758; 1:200), anti- Chromogranin A (rb, Immunostar, 20085; 1:200). Highly cross-adsorbed Alexa Fluor secondary antibodies (ThermoFisher; 1:500) were used. Nuclei were identified through DAPI staining (ThermoFisher). All images were collected and then quantified using a Nikon A1 confocal microscope and Nikon Elements software (v.5.30.05). The percentage of immune cell infiltration (insulitis) observed from immunofluorescence staining was scored as follows: none = no insulitis, moderate = infiltrate but preserved islet circumference, severe = infiltrate islet and loss of islet circumference. [000150] NanoString Spatial Proteomics: Paraffin embedded pancreata were used for Nanostring spatial proteomics analysis. Tissues were stained with morphology markers: AF-647 conjugated insulin (Cell Signaling; 9008s; 1:400) and nuclei marker (SYTO13). Tissues were hybridized using a pre-validated mouse GeoMx Immune cell panel (NanoString; GMX- PROCONCT-MICP) comprising of the following markers: CD45, CD4, CD11b, CD11c, CD3e, CD19, CD8a, CTLA4; housekeeping genes: Histone H3, S6, GAPDH; and IgG antibodies: Rb IgG, Rat IgG2a, and Rat IgG2b for background subtraction. All the markers were conjugated to unique UV-photocleavable oligos for indexing. At least 5-6 islets with insulitis were chosen as regions of interest (ROI) per mouse based on the morphology markers (insulin and nuclei). The ROIs were segmented into insulitic regions49. Oligos from the segmented ROIs were photocleaved, collected in a 96-well plate, and reads were counted using nCounter (Nanostring). Analysis was performed using the Nanostring software. Scaling was performed to normalize for any differences in tissue surface area and depth. After scaling, reads were normalized toMBHB Ref. No.24-0108-WO housekeeping markers and background was subtracted using IgG markers. Normalized counts were visualized as barplots using GraphPad Prism. [000151] Cell Mass and Insulitis Scoring: Pancreata were fixed in 4% paraformaldehyde, paraffin embedded, and sectioned at thickness of 5 mm. Three sections per mouse were used for analysis, with each section being spaced 100 mm apart. Tissue sections were immunostained with anti-insulin (ProteinTech; 15848-1-AP; 1:200) and followed by conjugated anti-rabbit Ig (Vector Laboratories) secondary antibody. A DAB (3,3’-diaminobenzedine) peroxidase substrate kit from Vector Laboratories was used for detection. After immunostaining, the tissue sections were counterstained with hematoxylin (Sigma). Images were acquired using a BZ-X810 fluorescence microscope (Keyence) and cell mass was determined by quantifying insulin+ area and whole pancreas area. Insulitis score reflects the degree of immune cells infiltration within pancreatic islets. The score system used as follow: 1 = no insulitis, 2 = infiltrate <50% circumference, 3 = infiltrate >50% circumference, 4 = infiltration within islet. [000152] Statistical Analyses: All data are represented as mean ±SEM. For comparisons involving more than two conditions, one-way ANOVA (with Tukey post-hoc test or Dunnett’s post-hoc test) was performed. GraphPad prism v10 was used for all statistical analysis and visualization. Statistical significance was assumed at p-value < 0.05. Example 1. Screening and optimization of core lipid LNP. [000153] To identify an optimized core LNP capable of efficient mRNA delivery, a library screening approach was utilized by establishing a library containing more than 60 LNPs. These LNPs comprise a lipid-grafted low-molecular-weight generation 0 PAMAM compound G0- dioleoylphospha-tidylethanolamine (DOPE), cholesterol, and 1,2-Distearoyl-sn- glycero-3-phosphoethanolamine-Polyethylene glycol (DSPE-PEG), with ratio of G0-C14 to DSPE-PEG is about 2:1 to about 15:1; cholesterol to DSPE-PEG is about 15:1 to about 40:1; and DOPE to DSPE-PEG is about 15:1 to about 30:1. The ethanol phase containing lipid components was mixed with the aqueous phase containing mRNA at a volume ratio of 1:3 to form LNP, Krüppel like factor 2 (KLF2) mRNA, an endothelial cell enriched transcription factor, was encapsulated into LNP. KLF2 direct downstream gene Rap guanine nucleotide exchange factor 3 (RAPGEF3) or endothelial nitric oxide synthase (eNOS3) was quantitatively analyzed by qPCRMBHB Ref. No.24-0108-WO to determine the KLF2 mRNA transfection efficiency on human primary microvascular endothelial cells (MVEC). [000154] After three rounds of screening, an optimized nanoparticle formulation (termed C9- LNP, G0-C14:DOPE:Cholesterol: DSPE-PEG2K=5:15:25:1, mol / mol) was identified that exhibits: (1) up to 99% mRNA encapsulation efficiency, (2) protection of mRNA from degradation by nucleases, (3) swift cellular uptake and endosomal escape to release cargo mRNA, (4) negligible immunogenicity and inflammation stimulation to host cells, (5) stabilized blood circulation, and (6) potent capability of target specific tissues or cell types by tailoring the surface chemistry via conjugating of targeting motifs (Figs.6A-6D). The optimized LNP enables potent mRNA transfection in MVEC in vitro and mice vasculature endothelium in vivo. Example 2. Characterization of V-CAM1 targeting lipid LNP. [000155] Previous studies over the last decade have focused on pathways and processes in cells that promote their antigenicity, including the polyamines / hypusine pathway77,81,82(and clinical trial NCT02384889) and the lipoxygenase pathway19,75,76. Because these pathways are ubiquitous in all cell types, cell-specific targeting of these pathways is necessary to avoid off- target or unwanted effects. This concept is also applicable to diseases where specific cell types or organs become dysfunctional. Notably, dysfunction of vascular endothelium contributes to perhaps more human diseases than any other tissue. Mechanisms of mechano-transduction are especially important in the vasculature. There, environmental mechanical stimuli provoke cellular responses in endothelial cells that induce an array of disorders, including atherosclerosis and acute and chronic pulmonary disease70,80,83,84. To therapeutically target endothelial responses, polymeric and lipid-based nanoparticles were engineered that carry a variety of nucleic acid cargo and are decorated with cell-targeting peptides for delivery in vivo. Three major forms of novel nanoparticles were engineered and formulated to display cell-targeting peptides and tailored to deliver different types of therapeutic nucleotides. Polyelectrolyte complex micelles deliver small RNAs (e.g. siRNAs, miRNAs for interference)26,71, lipids deliver functional mRNA (for protein expression), and fluorinated polyethyleneimine nanoparticles deliver large DNA fragments (e.g. plasmids for CRISPR gene editing)70,72,80. [000156] The present inventors previously generated nanoparticles that target inflamed vascular endothelium by displaying the VCAM1-binding peptide (VHPKQHR) (SEQ ID NO: 6)MBHB Ref. No.24-0108-WO on the nanoparticle surface. The VCAM1-targeting peptide was incorporated into the lipid (DSPE-PEG- VHPKQHR (SEQ ID NO: 6) in Fig.7A). VHPKQHR (SEQ ID NO: 6) was identified by phage display to specifically bind VCAM-1 in activated endothelium85. Transmission electron microscopy (TEM) images showed monodispersed VCAM1-targeting lipids with a diameter of ~70 nm when dry and a median hydrodynamic diameter of ~120 nm in deionized (DI) water (Fig.7A). VCAM1-targeting lipids delivered functional mRNA for mScarlet (a bright monomeric red fluorescent protein) to inflamed endothelial cells unlike the non-targeting lipid (Fig.7B). Studies in vivo (Fig.7C) demonstrated that these VCAM1- targeting lipids effectively deliver functional mScarlet mRNA to inflamed mouse lungs subjected to LPS, while non-targeting lipids showed limited potency of functional mRNA delivery. Example 3. In vitro lipid nanoparticle delivery of cargo to human -cells in vitro [000157] In initial studies to test the feasibility of using lipid-based nanoparticles to deliver cargo to human cells, self-assembled lipids were generated from a mixture of lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14)95, cholesterol, polyethylene glycol 2000 (PEG), 1, 2-Distearoyl-sn-glycero-3- phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), dioleoylphospha-tidylethanolamine (DOPE), and mScarlet mRNA. The nanoparticles were thenapplied to cultured human EndoC- H1 cells for 6 h. Figure 8 demonstrates that ~80% of cellsexpress functional mRNA for mScarlet. Further studies were then performed to test the feasibility of using GLP-1R targeted LNPs. GLP-1R, is enriched on cells in both human and mouse and exhibits limited tissue distribution86,87. Upon binding to ligand, GLP-1R exhibits internalization and membrane recycling88, thereby allowing incorporation of nanoparticle cargo. Self-assembled lipids that were functionalized with a previously described engineered peptide that interacts with GLP-1R (known as “eGLP1:” AibEGTFTSDVSSYLEEQAA- KEFIAWLVKGGPSSGAPPPSC-NH2 (SEQ ID NO: 1)) were generated. The LNPs were thenapplied to cultured human EndoC- H1 cells or human HEK293 cells for 4 and 18 h. Figure 9demonstrated that after 18 h ~80% of EndoC- H1 cells but not HEK293 expressed functionalmRNA for mScarlet. Example 4. Generation and characterization of LNPs with known peptides that interact with GLP-1RMBHB Ref. No.24-0108-WO [000158] In order to develop a -cell-specific LNP, an appropriate target on -cells had to be identified. One promising candidate is the GLP-1 receptor which is highly enriched on pancreatic -cells36. Previously, Knerr & Prakash, et al. used a human GLP-1 receptor agonist (deemed enhanced GLP-1, eGLP1) to deliver antisense oligonucleotides to -cells22. [000159] The present study focused on developing nanoparticles that are functionalized with known peptides that interact with GLP-1R and identifying new peptides that interact withNTPDase3, FXYD2 a, and GLP-1R by phage display. The functionalized nanoparticles werecomplexed with mScarlet-encoding mRNA to demonstrate delivery of this cargo to mouse and human cells in vitro (Fig.9 and Fig.14). An emphasis was placed on the testing of (a) cell lines from both mouse and human, and (b) primary mouse and human islets. Confirmation of successful, cell type-specific delivery were validated by immunofluorescence and flow cytometry (Figs.13A-13C), and additional studies of cell function (glucose stimulated insulin secretion), identity (qPCR for cell identity markers), and molecular pathways (by scRNA sequencing) were performed to interrogate if / how targeting and receptor engagement alters molecular pathways in the cell and possibly other islet cell types (Fig.18, Figs.19A-19B, and Fig.20). [000160] Self-assembled lipids were generated from a mixture of lipid grafted polyamidoamine (PAMAM) dendrimer (G0-C14)95, cholesterol, polyethylene glycol 2000 (PEG), 1, 2-Distearoyl- sn-glycero-3- phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), dioleoylphospha- tidylethanolamine (DOPE), and mScarlet mRNA. The lipid-PAMAM self-assembled into nanoparticles in aqueous solution and captures the mRNA by its cationic segments. GLP-1R- targeting peptides were conjugated to the PEG to be displayed on the nanoparticle surface. As demonstrated in the preliminary studies (Fig.9), early attempts demonstrated evidence oftargeting and delivery of cargo to cultured human cells at greater efficiency than non- cells.Functionalization of other peptides as identified from phage display screening followed similar / modified chemical approaches (see below). Nanoparticles were characterized by transmission electron microscopy using the FEI Spirit LaB6 Routine Electron Microscope with a voltage of 120 kV located in the Advanced Electron Microscopy Facility at The University of Chicago. The NMR spectrum was recorded by a 400 MHz NMR instrument. The hydrodynamic size and zeta potential was measured by Zetasizer Nano ZS90. [000161] ResultsMBHB Ref. No.24-0108-WO [000162] The human eGLP-1 peptide was conjugated onto an LNP core. This eGLP-1- functionalized LNP (Targeted LNP) exhibited a size of ~150 nm and measured a positive charge of ~27 mV (Figs.10A-10C). These results indicated that a stable, functionalized LNP that is poised to incorporate negatively-charged nucleic acid mRNA cargo has been generated. Example 5. Identification of with new peptides that interact with GLP-1R, NTPDase3, and FXYD2 a[000163] As noted, the primary goal of this proposal is to generate technology that optimizes cell-specific targeting of cells, independent of the nature of cargo. In this experiment, new peptides that engage GLP-1R and peptides that interact with the cell-specific proteins NTPDase358, and FXYD2 a56 were identified. To the inventors’ knowledge, no peptides have yet beenidentified that interact with NTPDase 3 and FXYD2 a. To identify peptides that bind to theseproteins, phage display was utilized by contract to Creative Biolabs. Phage display is an interactive selection technique in which a library of peptide variants (in our case 9-mers) is displayed on the surface of a virion. These virions undergo partitioning (panning) against theprotein of interest (GLP-1R, NTPDase 3, or FXYD2 a) in an iterative manner, followed by phageamplification (see

[0091] for a review). Creative Biolabs leveraged premade 9-mer peptide library. Creative Biolabs then validated and sequenced up to 40 clones. The sequences of these clones were aligned and consensus peptides were functionalized to nanoparticles and processed as mentioned above. Example 6. Testing of cell type-specific delivery of cargo in cell lines [000164] To initially test the nanoparticle cell-type delivery, they were cultured at variousconcentrations with human cell lines, of which at least two exhibited GLP-1R (EndoC- H1 cellsand HEK293 cells transfected with human GLP-1R) and two negative control cells (HEK293 cells and PANC1 human pancreatic ductal cells). For mouse, two cell lines (MIN6 and TC3 cells) and two negative control cells (NIH3T3 fibroblasts and TC1.6 cells) were used. Cells were incubated with nanoparticles for 0 or 6 h, washed, and imaged for mScarlet by fluorescence microscopy after 6, 12, 24, and 72h. Quantitation of uptake efficiency by the nanoparticles were performed by flow cytometry for mScarlet-pos vs. mScarlet-neg cells (Figs.13A and Fig.13B).MBHB Ref. No.24-0108-WO Example 7. In vitro liposomal nanoparticle delivery of Cd274 cargo to mouse and human cell lines to produce PD-L1 [000165] -cell centric therapies show promise in improving T1D outcomes13,15,17,18,19,, but off-target effects of systemic drug delivery pose a significant barrier to drug development. LNP technology offers a unique approach to mitigate off-target effects and focus therapeutic action to the islet. One potential therapeutic cargo to use in this system is mRNA encoding the immune checkpoint protein PD-L1 (Cd274). It is known that PD-L1, through interaction with the PD-1 receptor on immune cells, induces an immunosuppressive response99. In T1D, a subset of -cells express PD-L1, which interacts with PD-1 on T-cells to induce immunosuppression and escape from immune destruction47. Unfortunately, these few remaining -cells are unable to maintain glucose homeostasis, leading to continued diabetes19,47. Therefore, the -cell specificity of eGLP-1-LNP was confirmed and leveraged to expand PD-L1 expression on -cells. [000166] To test if the targeted LNPs can deliver functional mRNA in vitro, 300 ng of human mRNA encoding PD-L1 (Cd274) was incorporated into the LNP and used it to treatmouse (MIN6) and human (EndoC- H1) -cell lines. After overnight exposure to LNPs, PD-L1 production was quantified via fluorescence activated cell sorting (FACS) in MIN6 cells and observed that ~25% of cells exhibited PD-L1 expression on the cell surface, suggesting that the LNP-delivered mRNA was subsequently translated into protein (Figs. 12A-12B). Interestingly, PD-L1 expression was also observed in the non-targeted LNP condition, which supports previous observations that LNPs have an inherent ability to deliver cargo in a non-cell specific manner30. Additionally, after exposing targeted LNPs to EndoC- H1 cells for 1or 3 days, there was continued PD-L1 expression measured via western blot (Fig. 12C). These data demonstrate that prolonged exposure of mRNA cargo from LNPs produces stable PD-L1 at the cell surface. Importantly, these data suggest that the core LNP can target mouse and human -cell lines, while also delivering functionalized cargo. Example 8. Testing of cell type-specific delivery of cargo in primary islets [000167] For nanoparticle-peptide conjugates that showed cell specificity above, these conjugates were advanced to the study of primary human and mouse islets (from C57BL6 / J mice). Human islets were obtained from either the Integrated Islet Distribution Program (IIDP) orMBHB Ref. No.24-0108-WO the Alberta Diabetes Institute (ADI), as previously done19,89. Islets were treated intact with various concentrations of the selected nanoparticle conjugate carrying mScarlet mRNA under similar conditions as with the cell lines. At least 6 human islet donors (3 male and 3 female) were used for these studies. Following incubation, the following studies were performed: [000168] Immunofluorescence: Islet were embedded in agarose, immunostained for insulin, glucagon, somatostatin, DAPI and subjected to immunofluorescence for each of these proteins and mScarlet to visualize overlap of mScarlet with insulin(+) cells. [000169] Quantitation of cell type specificity: To quantitate colocalization of mScarlet with each of the islet cell types, flow cytometry of dispersed islets was performed, subsequently fixed and immunostained for each islet cell type and for mScarlet (using an mScarlet antibody). The percentage of mScarlet(+) / insulin(+) cells relative to other cell types that were mScarlet(+) were quantitated. The dispersion, immunostaining, and quantitation of human islets by this method was previously described89. [000170] cell functionality: These experiments were performed to interrogate if / how nanoparticle transduction impacts cell function. These experiments were important given that the nanoparticles engaged functional receptors on cells. Based on optimized conditions that allow for maximal transduction specifically of cells from the experiments above, both mouse and human islets were used from these conditions to perform perifusion analysis using the Biorep perifusion instrument. Figure 15 shows a perifusion profile of human islets, indicating intact first and second phase insulin secretion. The profile of transduced islets were compared with non- transduced islets. [000171] Gene expression analysis: These experiments were performed to interrogate the effect of nanoparticle transduction on intracellular pathways of cellular differentiation, stress signaling (e.g. MAPK, JNK, ER stress, oxidative stress), cell cycle / replication, and cell function (secretory pathways). Moreover, use of single cell sequencing allowed not only assess to cell pathways, but also how transduction by nanoparticles might influence the transcriptome of other islet cell types. Based on conditions that allow for maximal transduction specifically of cells, both mouse and human islets were used from these conditions to perform single-cell RNA sequencing. The inventors have previously extensively employed single cell RNA sequencing, and cell dispersion, use of the 10X Genomics platform, annotations of islet cell types, and bioinformatics analysis pipelines19,90.MBHB Ref. No.24-0108-WO Example 9. Express PD-L1 on human -cells [000172] While fluorescence is a good marker to test the feasibility of eGLP-1 LNP to target human -cells (Fig. 21), ideally, it was important to show that eGLP-1-LNP can deliver potentially therapeutic cargo to human -cells. The goal was to test that eGLP-1 targeting LNP can deliver Cd274 mRNA cargo that will be translated into PD-L1 onto the surface of human - cells. To test this goal, the following experiments were carried out using male and female non- diabetic donor human islets obtained from the Integrated Islet Distribution Program (IIDP) or the University of Alberta. [000173] PD-L1 focused histological analysis: Donor human islets were exposed in vitro to PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying Cd274 mRNA cargo (300 ng). PD-L1 takes about ~12 hours to appear at detectable levels to the cell membrane. Therefore, to allow for cell surface accumulation and protein detection, islets were treated with LNPs carrying Cd274 mRNA cargo for 8 hours, LNPs were washed out, and then waited for 72 hours. Islets were histologically processed for insulin, glucagon, somatostatin, and ghrelin and analyzed for any colocalization with PD-L1. [000174] Quantify PD-L1 expression on human -cells: Donor human islets were exposed to PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying Cd274 mRNA cargo for 8 hours in vitro. After 72 hours, FACS analysis was performed of isolated pancreatic islets and quantified overlap between insulin positive cells and PD-L1. Example 10. Functionalized nanoparticle deliver cargo to cells in vivo [000175] The goal of these experiments was to advance the most promising functionalized nanoparticles described in Examples 7, 8, and 9 (also see Figs.16A-16C, Fig.21, and Fig.22) into systems in vivo: normal mice, “humanized” mice (immunodeficient mice transplanted with human islets), and NOD mice. In NOD mice, PD-L1-encoding mRNA was delivered to cells to test for prevention / reversal of T1D. Subsequent analyses included tissue verification of -cell targeting (by immunostaining and flow cytometry of islets), intravital imaging of endogenous and transplanted human islets in live mice, and assessment of diabetes prevention / reversal. The experimental designs described below were utilized for each functionalized nanoparticle from Examples 7, 8, and 9 that demonstrated cell-specific targeting.MBHB Ref. No.24-0108-WO [000176] Although the previous examples support the notion that eGLP-1-LNPs target -cell lines and enriches mRNA delivery to the pancreas, the data do not address direct delivery to - cells in vivo. Therefore, we tested whether eGLP-1 LNPs target -cells. To test if the LNPs can deliver mRNA cargo specifically to the pancreas in vivo, LNPs carrying fluorescent mRNA cargo (Cy5-eGFP, 25μg) were injected into the intraperitoneal cavity of C57BL / 6J mice (Fig.16A). Five different conditions were used to compare Cy5-eGFP delivery: PBS, eGLP-1 LNPs without cargo (Empty LNPs), naked mRNA, non-targeted LNPs, and targeted eGLP-1 LNPs. [000177] For these experiments, C57BL6 / J mice (male and female) were injected into the peritoneal cavity (i.p.) with varying concentrations of functionalized nanoparticle with Cy5-eGFP encoding mRNA and euthanized 1 day post injection to visualize for Cy5 in cells. After overnight exposure and dissection of major organs, ex vivo assessment for fluorescent signal was performed using an in vivo imaging system (IVIS). Pancreata were harvested, fixed, paraffin- embedded, and stained for insulin, glucagon, and somatostatin, and visualized forinsulin(+) / mScarlet(+) (positive cells), insulin(-) / mScarlet(+) (positive non- cells), andinsulin(+) / mScarlet(-) (negative cells) cells to verify -cell targeting. Other organs (liver, fat, brain, spleen, etc.) were also harvested and analyzed for potential Cy5(+) cells (the finding of which would indicate lack of tissue-specific targeting). [000178] Pancreatic histological analysis: Mice were intraperitoneally (i.p.) injected with PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying fluorescent mRNA cargo (e.g.eGFP or mScarlet). After 24 hours, pancreatic tissue was collected and stained for insulin ( -cellmarker), glucagon ( -cell marker), somatostatin ( -cell marker), ghrelin ( -cell marker), andCela3b (acinar cell marker) and determined any colocalization with fluorescent protein expression (GFP or mScarlet). [000179] Quantify LNP targeting efficiency on -cells: FACS analysis of isolated pancreatic islets was performed and quantified overlap between insulin positive cells and fluorescent protein expressing cells. [000180] Quantify LNP targeting specificity on GLP-1R: To specifically test whether LNPs target -cells via GLP-1R mediated responses, islets were isolated from mice and GLP-1R was blocked with exendin-4, a known agonist of GLP-1R, before treatment with vehicle, untargeted LNP, and eGLP-1 targeting LNP. Isolated islets were pretreated with exendin-4 for 2 hours and then treated with LNPs for 24 hours (Fig.18, Figs.19A-19B, and Fig. 20).MBHB Ref. No.24-0108-WO [000181] Results [000182] Consistent with the in vitro data, some fluorescence was observed in the liver, pancreas, and spleen with the non-targeted LNP (Figs.16B-16C), which again supports the systemic capability of non-cell specific LNPs to deliver mRNA cargo30. However, with the targeted LNP, the highest fluorescence intensity was observed in the pancreas (Figs. 16B-16C). These data show that fluorescent cargo can be delivered to the mouse pancreas using targeted LNPs. Example 11. Functionalized nanoparticles delivered to human -cells in vivo [000183] Translating LNP therapeutics from the mouse to human is essential to bringing nanomedicine therapies to the clinic. Although NOD mouse models may recapitulate some aspects of T1D, these systems lack genetic heterogeneity and complexity that are present in human T1D patients102. Furthermore, islet architecture differs between the mouse and human whereby human islets display heterogeneous localization of -cells, whereas in the mouse islet - cells appear more along the periphery103. With these distinctions, it was important to confirm that the LNPs are feasible for use in human -cells. Example 7 (Fig.12C) suggested that the eGLP-1- LNP can deliver functionalized cargo to a human -cell line, but it remains to be seen if the LNP targets -cells within the human islet. Therefore, to further contribute knowledge about human - cell therapies and to create an exciting tool to target human -cells, we tested whether eGLP-1 functionalized LNP targets human -cells and delivers functional mRNA cargo (Example 9 and Fig. 21).[000184] Although the eGLP-1 LNP targets EndoC- H1 cells in monoculture, it is yet to bedetermined if it can target -cells within the human islet microenvironment. To test whether eGLP-1 functionalized LNP can deliver fluorescent mRNA cargo to human islets and enrich fluorescent protein expression in -cells, the following experiments using male and female non- diabetic donor human islets obtained from the Integrated Islet Distribution Program (IIDP) or the University of Alberta were used (Fig. 21). [000185] For these experiments, “humanized” mice were generated by transplanting human islets under the right kidney capsule prior to injection with functionalized nanoparticle withmScarlet-encoding mRNA. Human islets were obtained from nondiseased donors ( 30 y of age,BMI preferably <30). These islets were transplanted into the kidney capsule (1000-2000 IEq) ofMBHB Ref. No.24-0108-WONOD-SCID-IL2r -null (NSG) immunodeficient mice, as previously published

[0092] . After 1 weekof recovery following transplantation, mice were injected with functionalized nanoparticle (at a dose determined in above) with mScarlet-encoding mRNA. Mice were euthanized at 0, 3, 5, and 7 days post injection. Kidneys and pancreata from 3 mice per time point were harvested, fixed, paraffin-embedded, and stained for insulin, glucagon, and somatostatin, and visualized forinsulin(+) / mScarlet(+) (positive cells), insulin(-) / mScarlet(+) (positive non- cells), andinsulin(+) / mScarlet(-) (negative cells) cells to verify -cell targeting. Other organs (liver, fat, brain, spleen, etc.) were also harvested and analyzed mScarlet(+) cells, as noted above. [000186] Quantify LNP targeting efficiency on human -cells: Non-diabetic donor human islets were exposed in vitro to PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying fluorescent mRNA cargo (e.g. eGFP or mScarlet, 300 ng) for 8 hours. 24 hours after removing the LNP solution, FACS analysis of islets was performed and quantifications were compared between insulin positive cells, non-insulin positive cells, and fluorescent protein expressing cells. [000187] Histological Analysis: After following the same treatment protocol above (Quantify LNP targeting efficiency on human -cells), human islets were histologically processed and stained for endocrine markers including insulin, glucagon, somatostatin, and ghrelin to determine any colocalization with fluorescent protein expression (GFP or mScarlet). Example 12. Prevention of diabetes in NOD mice using targeted delivery of PD-L1 [000188] Data disclosed herein above suggest that eGLP-1 functionalized LNP delivers Cd274 mRNA and produces stable PD-L1 on the surface of mouse and human cell lines. However, it was unclear if Cd274 delivery and subsequent production of PD-L1 preserves -cell mass, alters insulitis, or delays T1D progression. Therefore, the goal is to test whether eGLP-1 LNPs with Cd274 mRNA cargo delay T1D progression, preserve -cell mass, and reduce insulitis in vivo. To test the possibility for efficacy of cargo delivery in vivo, the potential of therapeutics using the functionalized nanoparticles was tested by performing the following experiments in a mouse model of T1D (non-obese diabetic, or NOD, mice) starting at 6 weeks of age (Fig.17, Fig 22, and Fig.26A-26I). [000189] Six-week-old female prediabetic NOD mice were injected i.p. with functionalized nanoparticle with (HA)-PD-L1-encoding mRNA in a prevention model. For these prevention studies, an “n” of 15 NOD animals per group for the mouse studies was proposed: if 80% ofMBHB Ref. No.24-0108-WO female NOD mice spontaneously developed diabetes between age 12-24 weeks, then a sample size of 15 per group has a 95% power to detect an increase in survival proportion of 0.66 with a significance level (alpha) of 0.05. The frequency of injections was determined empirically based on the persistence of mScarlet protein in the above experiments. Briefly, the following outcomes were measured. [000190] PD-L1 focused pancreatic histological analysis: Mice were i.p. injected with PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying Cd274 mRNA cargo (25 μg). Previous data suggests that PD-L1 trafficking to the membrane takes at least 12 hours100. Furthermore, previous experience suggested that multiple injections of therapeutic cargo are necessary to elicit a phenotype. Therefore, to sufficiently expose -cells to adequate amounts of Cd274 mRNA cargo, PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying Cd274 mRNA cargo were i.p. injected twice weekly into NOD mice at 6 weeks old until 8 weeks old. Pancreatic tissuewere histologically processed for insulin ( -cell marker), glucagon ( -cell marker), somatostatin( -cell marker), ghrelin ( -cell marker), and Cela3b (acinar cell marker) and analyzed for anycolocalization with PD-L1. [000191] Diabetes incidence and glycemic control: Blood glucose was measured on the cut tail 1-2 times a week. Local anesthesia was provided after measurement. Mice were considered as having diabetes after 2 consecutive blood glucose readings above 300 mg / dL [19,76,78]. Incidence was analyzed at the endpoint at 25 weeks of age. [000192] Analysis of pancreatic immunohistochemistry and morphometry: Pancreata of NOD mice (8 weeks old) at prediabetic stages, diabetes, and non-diabetes end-stage injected with PBS, non-targeted LNPs, and eGLP-1 targeted LNPs carrying Cd274 mRNA cargo were harvested, weighed, fixed, paraffin-embedded, and stained for insulin and glucagon to estimate relative islet area and total cell mass, as we have done previously [19,76]. [000193] PD-L1 tissue immunostaining: Additionally, pancreata were stained for the hemagglutinin tag (HA-PD-L1) (to distinguish from endogenous PD-L1) and insulin to ensure appropriate targeted delivery of PD-L1. [000194] Biodistribution, pharmacokinetics, tolerable doses, toxicity and immunogenicity of the targeted nanoparticles: Detailed protocols were listed in a previous study

[0026] . Preliminary results demonstrated that four injections over the course of two weeks starting at 6 weeks of ageMBHB Ref. No.24-0108-WO until 8 weeks of age of Cd274 mRNA-encapsulated LNPs (1 mg mRNA / kg body weight) (Fig. 17, Fig.22) had no toxicity in major organs and do not trigger immunogenicity in NOD mice. [000195] Insulitis scoring: Pancreata of mice at early prediabetic stages (8 weeks old) will be harvested, weighed, and histologically processed for insulin. The degree of mononuclear infiltrate will be assessed by immunohistochemistry, and insulitis will be scored using the following system: 1) no insulitis, 2) infiltrate < 50% circumference, 3) infiltrate > 50% circumference, and 4) infiltration within islet. Example 13. Characteristics of eGLP-1 Conjugated Lipid Nanoparticles [000196] To achieve specific delivery of mRNA cargo to pancreatic -cells, a -cell receptor targeting peptide was attached onto LNPs. As previously described, the LNP core consists of a lipid-modified low-molecular-weight poly(amidoamine) (PAMAM) compound G0-C14, 1,2- Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), cholesterol, and 1,2-Distearoyl-sn- glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG). Glucagon-like peptide-1 receptor (GLP-1R) is highly enriched on pancreatic -cells36and has a well-characterized ligand, GLP-137. To leverage this receptor / ligand interaction, a synthesized enhanced peptide version of GLP-134,35(known as eGLP-1) was conjugated via click chemistry to the core LNP. The LNP core conjugated with eGLP-1 (hereafter referred to as eGLP-LNPs or conjugated LNPs) was compared with the unconjugated LNPs. Using transmission electron microscopy imaging, it was observed that eGLP-LNPs were significantly larger than unconjugated LNPs (Fig.23A). Dynamic light scattering indicated that eGLP-LNPs were ~75 nm in size, while LNPs were ~60 nm (Fig. 23B). Size distribution analysis peaks were consistent with these size measurements, indicating that conjugation with the eGLP peptide increased the size of LNPs (Fig.23C). To assess the charge of the LNPs, zeta potential measurement analysis was performed. Both conjugated and unconjugated LNPs had a positive charge of ~20 millivolts (mV) (Fig. 23D), indicating the potential for increased stability of negatively charged cargo within these LNPs38. Lastly, a RiboGreen (RNA detection) assay showed both eGLP-LNPs and unconjugated LNPs displayed ~99% mRNA encapsulation efficiency (Fig.23E). These data indicated that the addition of the eGLP-1 peptide did not impair LNP encapsulation efficiency. [000197] To verify that the eGLP-LNP displayed downstream GLP-1R activity consistent with engagement of eGLP-1 with the GLP-1R, a G-protein-coupled receptor (GPCR) internalizationMBHB Ref. No.24-0108-WO assay (Fig.23F) was utilized. The U2OS cell line designed for chemiluminescence detection upon internalization of GLP-1R with ligand binding39,40was used. As expected, incubation of this cell line with the ligands exendin-4 or eGLP-1 elicited GLP1R internalization as evidenced by increases compared to vehicle controls (Fig.23G). Whereas unconjugated LNPs did not elicit a GLP1R internalization, eGLP-LNPs increased luminescence activity (Fig.23G), indicating that the eGLP-1 targeting peptide engages with GLP-1R even when conjugated to the LNP core. Taken together, these data confirm the successful generation of eGLP-LNPs with the potential for -cell targeting. Example 14. eGLP-LNPs Enrich Cargo Delivery to Mouse cells [000198] Next, the -cell specificity of eGLP-LNPs in mouse -cells in vitro was evaluated. Isolated mouse islets were incubated overnight with eGLP-LNPs or unconjugated LNPs carrying the fluorescent compound Cy5, and fluorescence distribution among islet cell types was quantified by flow cytometry. Notably, it was observed that incubation of islets with eGLP-LNPs resulted in significantly greater enrichment of Cy5 within -cells (insulin-positive population) compared to unconjugated LNPs (~9% of compared to ~4%, respectively; P-value < 0.0001) (Fig.23H). Within -cells (glucagon-positive population), ~2% Cy-5 uptake was observed withboth eGLP-LNPs and unconjugated LNPs (Fig. 23I). Similarly, in all non- -cell types (totalinsulin-negative population, which includes -cell, -cell, PP-cells, and non-endocrine cells), Cy- 5 fluorescence delivery was <3% for both eGLP-conjugated and unconjugated LNPs, with the former showing a slight discernible fluorescence signal compared to untreated islets (Fig.23J). Collectively, these findings indicate that LNPs exhibit preferential delivery of cargo to -cells in isolated mouse islets general but that eGLP-LNPs show greater cargo delivery to -cells compared to unconjugated LNPs. [000199] Next, competition assays was performed using the high-potency GLP1R agonist exendin-4 (Ex-4)41,42in vitro. Isolated mouse islets were pretreated with Ex-4 for 1 hour and then exposed to Ex-4 and LNPs overnight (Fig.23H). Treatment with Ex-4 at 1 nM or 10 nM decreased the percentage of Cy5-positive -cells with eGLP-LNP treatment (Fig.23I). Ex-4 treatment did not affect the percentage of Cy-5-positive -cells seen upon exposure to unconjugated LNP, suggesting that the presence of the eGLP1 peptide on LNPs is required to elicit a block by Ex-4. When -cells were examined, there was an expected small increase inMBHB Ref. No.24-0108-WO fluorescence with both unconjugated and conjugated LNPs compared to untreated controls (Fig. 23J). However, Ex-4 treatment induced no changes in the percent of -cells exhibiting Cy5 fluorescence (Fig. 23J). Collectively, these data suggest that eGLP-LNPs require GLP-1R to augment cargo delivery to -cells. Example 15. Delivery and uptake of cargo by eGLP-LNP into C57BL / 6J mouse pancreas in vivo [000200] Next, the ability of targeted LNPs to localize to the pancreas and other organs in vivo was test. Several control and experimental conditions were employed using an intraperitoneal (IP) delivery approach in 8-week-old C57BL6 / J mice that included phosphate-buffered saline (PBS) alone, eGLP-LNP with no cargo, Cy5 alone, unconjugated LNPs containing Cy5 cargo, and eGLP-LNPs containing Cy5 cargo. At 18 hours following IP injection, mice were euthanized and pancreas, liver, and other major organs were harvested and subjected to whole-organ fluorescence imaging (Fig.24A). No significant Cy5 fluorescence was observed in any organ with the PBS injection control and eGLP-LNP (no cargo) (Fig.24A & 24B, Fig 27A), and detectable but minimal fluorescence within the pancreas was observed with Cy5 alone (Fig. 24B). Both unconjugated LNP and eGLP-LNP delivered Cy5 to the pancreas, as demonstrated by enrichment in average radiant efficiency normalized to PBS (Fig.24A) but was greater with the eGLP-LNP (Fig.24B). Delivery of Cy5 to the liver was observed with the unconjugated LNP with much lower intensity observed with the eGLP-LNP (Fig.24A); notably, the ratio of pancreas-to-liver intensity was greater with the eGLP-LNP compared to unconjugated LNP (ANOVA P=0.0038) (Fig.2C), suggesting that the conjugation with the eGLP-1 peptide enhanced pancreas specificity. [000201] To assess the -cell specificity of cargo delivery by LNPs in vivo, -cells were ablated in male C57BL / 6J mice using a single high dose of the -cell toxin streptozotocin (STZ)43. At 48 h after STZ injection, the body weights of the mice were reduced compared to water injected controls, and random-fed blood glucose levels were greater than 500 mg / dL on average, consistent with mice developing diabetes from ablation of -cells (Figs.27B and 27C). Following -cell ablation, mice were injected with LNPs loaded with Cy5 fluorescent cargo or controls to determine organ biodistribution. As expected, PBS and naked Cy5 IP injection led to minimal fluorescence in all major organs (Figs. 24D and 24E). Whereas the unconjugated andMBHB Ref. No.24-0108-WO eGLP-LNPs induced measurable fluorescence in the pancreas, liver, and spleen, the eGLP-LNP injections showed significantly less fluorescence in these tissues (Figs.24D and24 E, Fig.27D). Notably, the ratio of pancreas-to-liver intensity was not distinguishable between unconjugated LNPs and eGLP-LNPs (Fig.24F). Collectively, these data suggest that the enriched pancreas uptake of Cy5 by eGLP-LNPs requires the presence of -cells. Example 14. Pancreas-specific delivery of cargo with eGLP-LNP in NOD mice [000202] The non-obese diabetic (NOD) mouse is an extensively-characterized preclinical model of T1D44, where early insulitis and intercellular communication between -cells and the immune system give way to later -cell destruction1. To test whether eGLP-LNPs can deliver Cy5 to -cells in this model as a prelude to eventual therapeutic cargo delivery, 8 and 9-week-old female NOD mice were injected with LNPs or controls, as performed previously, and 18 h later, organs were collected for fluorescence imaging. As expected, PBS injections showed little or no fluorescence uptake in tissues (Fig. 27H). However, unlike age-matched C57BL6 / J mice, no statistical difference was observed in pancreas fluorescence between eGLP-LNPs and unconjugated LNPs (Fig.27I). This unexpected result led the inventors to consider if the invading insulitis in this strain may reduce or suppress the expression of GLP-1R, thereby diminishing the specificity of eGLP-LNPs. [000203] To gain insight into GLP-1R expression changes in NOD mice, single-cell RNA sequencing (scRNA-seq) data from a mouse islet atlas45were first re-analyzed. In NOD islets, the notion that -cells had the highest level of Glp1r expression compared to other cell types36(Fig.27E) was supported. Furthermore, Glp1r transcript levels were highest in 5-week-old NOD mice but decreased significantly over time (Fig. 27F), suggesting that GLP-1R protein levels decline with age and advancing insulitis. To verify these Glp1r expression data at the protein level, immunofluorescence was performed for GLP-1R in NOD mouse pancreas at 6, 8, 10, and 12 weeks of age (Fig.25A). GLP-1R expression decreased significantly with age (Fig.25B), although islet-to-islet heterogeneity was observed. It was surmised that the heterogeneity in GLP-1R immunostaining correlates to the degree of insulitis, the frequency and severity of which increase with age in NOD mice. Upon delineation of GLP-1R expression in islets by severity of insulitis, a statistically significant inverse correlation was observed between severity of insulitis and GLP-1R expression that was independent of age (Fig.25C-F, Fig.27G). BecauseMBHB Ref. No.24-0108-WO the frequency of insulitis of any degree is lowest at 6 weeks of age (Fig.25C, Fig. 27G), it was conjectured that cargo delivery to -cells would be more effective beginning at 6 weeks of age. As observed previously, control injections (PBS and Cy5 alone) displayed little to no fluorescence in all major organs in NOD mice injected at 6 weeks of age (Figs.25G and 25H, Fig.27J). Mice injected with unconjugated LNPs carrying Cy5 showed little uptake and by contrast, NOD mice injected with targeted LNP with Cy5 displayed increased fluorescence in the pancreas and liver (Fig. 25G). As observed previously within C57BL / 6J, the ratio of pancreas-to- liver intensity was greater with the eGLP-LNP compared to unconjugated LNP (ANOVA P=0.0044) (Fig.25I). These data show that eGLP-LNPs can be utilized in a pre-clinical model of T1D at an appropriate age before disease onset. Example 15. Delivery of Therapeutic mRNA with LNPs In Vitro and In Vivo [000204] To explore the utility of LNPs as a therapeutic tool in T1D, Cd274 mRNA was encapsulated, which encodes for the immune checkpoint protein PD-L1. PD-L1 is expressed on the surface of many epithelial cell types, where it binds to its cognate receptor programmed cell death protein-1 (PD-1) on adaptive immune cells, leading to an inhibitory signal that suppresses the immune response46. During T1D progression, PD-L1 expression has been shown to increase in a subset of -cells that evade T-cell mediated insulitis and cell death, and its augmentation on -cells can lead to T1D delay19,47-49. Therefore, increasing PD-L1 expression on -cells with LNPs could attenuate the immune response and preserve -cell function. To ensure that LNPs loaded with Cd274 mRNA can deliver PD-L1 protein, MIN6 -cells were exposed to LNPs for 18 h, then performed flow cytometry to ensure that protein is expressed on the cell surface (Fig. 26A). Furthermore, exposure of MIN6 cells to eGLP-LNPs loaded with Cd274 mRNA resulted in cell surface PD-L1 production that persisted for up 5 days (Fig. 28A). Similar to the results from fluorescence cargo delivery, these data show that LNPs deliver cargo in vitro to mouse cells and that Cd274 mRNA cargo is successfully translated into PD-L1. [000205] Next, to evaluate the ability of LNPs to deliver functional PD-L1 to -cells in NOD mice, pre-diabetic 6-week-old NOD mice were injected with controls (PBS injections or eGLP- LNPs containing no cargo) or LNPs carrying Cd274 mRNA twice weekly for two weeks (Fig. 26B). At the end of the treatment period, diabetes incidence was recorded as well as the pancreas was collected to determine cellular localization of PD-L1. Approximately 70% of the miceMBHB Ref. No.24-0108-WO treated with eGLP-LNPs with Cd274 mRNA remained diabetes-free, compared to 40% and 30% with eGLP-LNPs containing no cargo and PBS injections respectively (Fig.26C). When examining cellular localization, compared to control injections, the unconjugated LNPs and eGLP-LNPs delivered Cd274 mRNA to produce PD-L1 on the surface of islets (Fig.26D). To examine targeting specificity, PD-L1 expression in the islet environment was examined. By comparing PD-L1 and insulin double-positive cells to overall insulin-positive cells, it was observed that eGLP-LNPs were more selective in delivering to insulin-positive cells, compared to untargeted LNPs that led to broad expression of PD-L1 (Fig. 26E). PD-L1 fluorescent intensity was also quantified within the acinar area, and there were no differences between conditions which suggested that LNP delivery was specific to the endocrine compartment (Fig. 26F). [000206] To determine if LNP-induced PD-L1 expression on -cells successfully dampens the islet autoimmune response, insulitis scoring of the islet area and spatial proteomics with Nanostring® were performed using an immune cell panel on the insulitic regions. Notably, - cell mass showed an increasing trend with LNP-induced PD-L1 expression (Figs.26G and 26H). Furthermore, eGLP-LNP treated islets displayed a decrease in severe insulitis compared to PBS, empty LNP, and untargeted LNP + Cd274 conditions (Fig.26I). Nanostring® data of the insulitic regions indicated no change across all conditions, suggesting that the LNP effects were localized to islet areas and not seen in immune populations (Fig.28B). These data suggest a slight protective response that is specific to -cells. [000207] Discussion [000208] Although T1D is viewed primarily as a disorder of immune tolerance leading to autoimmunity against islet cells, the results of immune targeting therapies have shown limited effects on either the prevention or reversal of the disease2-4. These findings have raised the need to consider combination immunotherapies targeting multiple facets of the immune response11,12. More recently, it has been appreciated that non-immune cell types in the pancreas, especially cells, might contribute to T1D pathogenesis by presenting antigens that initiate or augment the autoimmune response1. These findings suggest that combination therapies targeting the immune system and other pancreatic cell types might hold greater promise for therapy. However, the targeting of cells for drug delivery represents a challenge because of the relatively rare population of such cells and due to the lack of ideal vehicles for delivering cell therapeutics.MBHB Ref. No.24-0108-WO LNPs have emerged as promising vehicles for cell-specific drug delivery and have been safely and effectively used to deliver immune-sensitizing cargo for vaccines or cancer therapy25. In this study, a formulation of LNPs conjugated to a peptide (eGLP-1) that binds a receptor (GLP-1R) enriched on cells were utilized. The findings demonstrate (a) evidence that the conjugated LNPs interact with the membrane-bound GLP-1R and is subsequently internalized, (b) an enrichment of mRNA cargo delivery to cells following delivery of conjugated LNPs to mice in vivo, and (c) evidence that cargo delivery to cells have the potential to modify disease pathogenesis. [000209] Cell surface receptors on nanoparticles are an effective tool for achieving cell-specific targeting of therapeutics. By identifying and utilizing distinct receptor profiles, enhanced targeting can selectively guide therapeutic agents to minimize off-target effects and enhance treatment efficacy. Previous studies have demonstrated the feasibility of targeting peptides to deliver cargo within the lung, liver, immune cells, and other organs50–54. In the pancreas, cell-specific targeting has been achieved by conjugation of GLP-1R ligands to estrogen, antisense oligonucleotides (ASOs), and a dual-anchor coupling strategy21–23,34. The present study further supports the utility of GLP-1R ligands for -cell targeting but also suggests GLP-1-conjugated LNPs alone cannot modulate insulitis in NOD mice. Prior studies have demonstrated that the GLP-1 analog Ex-4 can reduce insulitis and delay diabetes onset when administered to NOD mice55. Although GLP-1R becomes internalized upon engagement with eGLP-LNPs, the present study suggests a beneficial effect if receptor activation is coupled with mRNA cargo. Further expanding the targeting repertoire to other -cell-enriched proteins, such as NTPDase3, DPP6, andFXYD2 a, could augment or improve -cell therapeutics. However, no binding peptides tothese other proteins currently exist that would enable functionalization to LNPs. [000210] LNP technology represents a promising strategy for improving T1D treatment by enhancing the expression of key -cell molecules that modulate autoimmune responses. Previous studies have demonstrated the protective effects of molecules such as CTLA4, BCL-XL, and IL- 10 in -cells, which can mitigate immune-mediated damage59–61. PD-L1 is a particularly promising immune checkpoint protein in the setting of T1D. The interaction between -cell PD- L1 and its receptor PD-1 on immune cells is a critical mechanism for dampening autoimmune attack in the context of T1D treatment and islet transplantation19,47,48,62,63. In the present study, the delivery of PD-L1 mRNA to -cells via eGLP-LNPs led to dampened diabetes incidence and reduced severe insulitis, indicating that targeted delivery can modulate local immune responses.MBHB Ref. No.24-0108-WO Spatial proteomics analysis of insulitic regions showed no changes in PD-L1 expression in surrounding immune cells, suggesting that PD-L1 upregulation was restricted to -cells, where it exerted a localized effect on the autoimmune process. This -cell-specific modulation highlights the therapeutic potential of LNP-mediated delivery in selectively altering immune interactions at the site of pathology. Aside from Cd274, other immune-regulatory mRNA cargo, such as cytokines IL-12 and IL-2364, or -cell transcriptional factors, such as PAX6 or MAF-Bmay offer further opportunities to enhance -cell survival and function in T1D. [000211] The embodiments illustratively described herein suitably can be practiced in the absence of any element or elements, limitation or limitations that are not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments claimed. Thus, it should be understood that although the present description has been specifically disclosed by embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of these embodiments as defined by the description and the appended claims. Although some aspects of the present disclosure can be identified herein as particularly advantageous, it is contemplated that the present disclosure is not limited to these particular aspects of the disclosure. [000212] Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. [000213] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in anyMBHB Ref. No.24-0108-WO other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. [000214] It should it be understood that, in general, where the disclosure, or aspects of the disclosure, is / are referred to as comprising particular elements and / or features, certain embodiments of the disclosure or aspects of the disclosure consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein. SequencesMBHB Ref. No.24-0108-WOMBHB Ref. No.24-0108-WO References 1. Atkinson MA, Mirmira RG. The pathogenic “symphony” in type 1 diabetes: A disorder of the immune system, cells, and exocrine pancreas. Cell Metab.2023;35(9):1500-1518. doi:10.1016 / j.cmet.2023.06.018 2. Pescovitz MD, Greenbaum CJ, Krause-Steinrauf H, et al. Rituximab, B-Lymphocyte Depletion, and Preservation of Beta-Cell Function. N Engl J Med.2009;361(22):2143-2152. doi:10.1056 / NEJMOA0904452 / SUPPL_FILE / NEJM_PESCOVITZ_2143SA1.PDF 3. Orban T, Bundy B, Becker DJ, et al. Co-stimulation modulation with abatacept in patients with recent-onset type 1 diabetes: A randomised, double-blind, placebo-controlled trial. The Lancet.2011;378(9789):412-419. doi:10.1016 / S0140-6736(11)60886-6 4. Herold KC, Bundy BN, Long SA, et al. An Anti-CD3 Antibody, Teplizumab, in Relatives at Risk for Type 1 Diabetes. N Engl J Med.2019;381(7):603-613. doi:10.1056 / NEJMOA1902226 / SUPPL_FILE / NEJMOA1902226_DATA-SHARING.PDF 5. Veld PI. Insulitis in human type 1 diabetes: A comparison between patients and animal models. Semin Immunopathol.2014;36(5):569-579. doi:10.1007 / S00281-014-0438- 4 / FIGURES / 1 6. Campbell-Thompson M, Fu A, Kaddis JS, et al. Insulitis and -Cell Mass in the Natural History of Type 1 Diabetes. Diabetes. 2016;65(3):719-731. doi:10.2337 / DB15-0779 7. Evans-Molina C, Sims EK, DiMeglio LA, et al. Cell dysfunction exists more than 5 years before type 1 diabetes diagnosis. JCI Insight.2018;3(15). doi:10.1172 / JCI.INSIGHT.120877 8. Buitinga M, Callebaut A, Sodré FMC, et al. Inflammation-Induced Citrullinated Glucose- Regulated Protein 78 Elicits Immune Responses in Human Type 1 Diabetes. Diabetes. 2018;67(11):2337-2348. doi:10.2337 / DB18-0295 9. Gonzalez-Duque S, Azoury ME, Colli ML, et al. Conventional and Neo-antigenic Peptides Presented by Cells Are Targeted by Circulating Naïve CD8+ T Cells in Type 1 Diabetic and Healthy Donors. Cell Metab.2018;28(6):946-960.e6. doi:10.1016 / J.CMET.2018.07.007 10. Marre ML, McGinty JW, Chow IT, et al. Modifying enzymes are elicited by ER stress, generating epitopes that are selectively recognized by CD4 + T cells in patients with type 1 diabetes. Diabetes. 2018;67(7):1356-1368. doi:10.2337 / DB17-1166 / - / DC1MBHB Ref. No.24-0108-WO 11. Roep BO, Thomaidou S, Tienhoven R van, Zaldumbide A. Type 1 diabetes mellitus as a disease of the -cell (do not blame the immune system?). Nat Rev Endocrinol 2020173. 2020;17(3):150-161. doi:10.1038 / s41574-020-00443-4 12. Long SA, Speake C. Combination therapy in recent-onset type 1 diabetes. Lancet Diabetes Endocrinol.2021;9(4):191-193. doi:10.1016 / S2213-8587(21)00044-9 13. Gitelman SE, Bundy BN, Ferrannini E, et al. Imatinib therapy for patients with recent- onset type 1 diabetes: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Diabetes Endocrinol.2021;9(8):502-514. doi:10.1016 / S2213-8587(21)00139-X 14. Herrath M von, Bain SC, Bode B, et al. Anti-interleukin-21 antibody and liraglutide for the preservation of -cell function in adults with recent-onset type 1 diabetes: a randomised, double-blind, placebo-controlled, phase 2 trial. Lancet Diabetes Endocrinol.2021;9(4):212-224. doi:10.1016 / S2213-8587(21)00019-X 15. Forlenza GP, McVean J, Beck RW, et al. Effect of Verapamil on Pancreatic Beta Cell Function in Newly Diagnosed Pediatric Type 1 Diabetes: A Randomized Clinical Trial. JAMA. 2023;329(12):990-999. doi:10.1001 / JAMA.2023.2064 16. Rosselot C, Li Y, Wang P, et al. Harmine and exendin-4 combination therapy safely expands human cell mass in vivo in a mouse xenograft system. Sci Transl Med. 2024;16(755):3456. doi:10.1126 / SCITRANSLMED.ADG3456 17. Engin F, Yermalovich A, Ngyuen T, et al. Restoration of the unfolded protein response in pancreatic cells protects mice against type 1 diabetes. Sci Transl Med. 2013;5(211). doi:10.1126 / SCITRANSLMED.3006534 / SUPPL_FILE / 5-211RA156_SM.PDF 18. Thompson PJ, Shah A, Ntranos V, Gool FV, Atkinson M, Correspondence AB. Targeted Elimination of Senescent Beta Cells Prevents Type 1 Diabetes. Cell Metab.2019;29:1045- 1060.e10. doi:10.1016 / j.cmet.2019.01.021 19. Piñeros AR, Kulkarni A, Gao H, et al. Proinflammatory signaling in islet cells propagates invasion of pathogenic immune cells in autoimmune diabetes. Cell Rep. 2022;39(13):111011. doi:10.1016 / J.CELREP.2022.111011 20. Ghosh K, Kanapathipillai M, Korin N, McCarthy JR, Ingber DE. Polymeric nanomaterials for islet targeting and immunotherapeutic delivery. Nano Lett.2012;12(1):203- 208. doi:10.1021 / NL203334C / SUPPL_FILE / NL203334C_SI_001.PDFMBHB Ref. No.24-0108-WO 21. Ämmälä C, Drury WJ, Knerr L, et al. Targeted delivery of antisense oligonucleotides to pancreatic beta-cells. Sci Adv. 2018;4:3386-3403. http: / / advances.sciencemag.org / 22. Knerr L, Prakash TP, Lee R, et al. Glucagon like Peptide 1 Receptor Agonists for Targeted Delivery of Antisense Oligonucleotides to Pancreatic Beta Cell. J Am Chem Soc. 2021;143(9):3416-3429. doi:10.1021 / JACS.0C12043 / ASSET / IMAGES / LARGE / JA0C12043_0010.JPEG 23. Wang S, Zhang Y, Wang Y, et al. An in situ dual-anchoring strategy for enhanced immobilization of PD-L1 to treat autoimmune diseases. Nat Commun 2023141.2023;14(1):1- 13. doi:10.1038 / s41467-023-42725-1 24. Hajj KA, Whitehead KA. Tools for translation: non-viral materials for therapeutic mRNA delivery. Nat Rev Mater 2017210.2017;2(10):1-17. doi:10.1038 / natrevmats.2017.56 25. Tenchov R, Bird R, Curtze AE, Zhou Q. Lipid Nanoparticles from Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement. ACS Nano. 2021;15(11):16982-17015. doi:10.1021 / ACSNANO.1C04996 / ASSET / IMAGES / MEDIUM / NN1C04996_0026.GIF 26. Zhou Z, Yeh CF, Mellas M, et al. Targeted polyelectrolyte complex micelles treat vascular complications in vivo. Proc Natl Acad Sci U S A.2021;118(50):e2114842118. doi:10.1073 / PNAS.2114842118 / SUPPL_FILE / PNAS.2114842118.SAPP.PDF 27. Kularatne RN, Crist RM, Stern ST. The Future of Tissue-Targeted Lipid Nanoparticle- Mediated Nucleic Acid Delivery. Pharmaceuticals. 2022;15(7). doi:10.3390 / PH15070897 28. Marques AC, Costa PC, Velho S, Amaral MH. Lipid Nanoparticles Functionalized with Antibodies for Anticancer Drug Therapy. Pharm 2023 Vol 15 Page 216.2023;15(1):216. doi:10.3390 / PHARMACEUTICS15010216 29. Shmeeda H, Amitay Y, Gorin J, et al. Coencapsulation of alendronate and doxorubicin in pegylated liposomes: a novel formulation for chemoimmunotherapy of cancer. J Drug Target. 2016;24(9):878-889. doi:10.1080 / 1061186X.2016.1191081 30. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 2021612.2021;6(12):1078-1094. doi:10.1038 / s41578-021-00358-0 31. Thomas SJ, Moreira ED, Kitchin N, et al. Safety and Efficacy of the BNT162b2 mRNA Covid-19 Vaccine through 6 Months. N Engl J Med.2021;385(19):1761-1773. doi:10.1056 / NEJMOA2110345 / SUPPL_FILE / NEJMOA2110345_DATA-SHARING.PDFMBHB Ref. No.24-0108-WO 32. Weber JS, Carlino MS, Khattak A, et al. Articles Individualised neoantigen therapy mRNA-4157 (V940) plus pembrolizumab versus pembrolizumab monotherapy in resected melanoma (KEYNOTE-942): a randomised, phase 2b study. www.thelancet.com. 2024;403:2024. doi:10.1016 / S0140-6736(23)02268-7 33. Melamed JR, Yerneni SS, Arral ML, et al. Ionizable lipid nanoparticles deliver mRNA to pancreatic cells via macrophage-mediated gene transfer. Sci Adv.2023;9(4). doi:10.1126 / SCIADV.ADE1444 / SUPPL_FILE / SCIADV.ADE1444_SM.PDF 34. Finan B, Yang B, Ottaway N, et al. Targeted estrogen delivery reverses the metabolic syndrome. Nat Med.2012;18(12):1847. doi:10.1038 / NM.3009 35. Sachs S, Bastidas-Ponce A, Tritschler S, et al. Targeted pharmacological therapy restores -cell function for diabetes remission. Nat Metab 202022. 2020;2(2):192-209. doi:10.1038 / s42255-020-0171-3 36. Campbell JE, Drucker DJ. Pharmacology, Physiology, and Mechanisms of Incretin Hormone Action. Cell Metab.2013;17(6):819-837. doi:10.1016 / J.CMET.2013.04.008 37. McLean BA, Wong CK, Campbell JE, Hodson DJ, Trapp S, Drucker DJ. Revisiting the Complexity of GLP-1 Action from Sites of Synthesis to Receptor Activation. Endocr Rev. 2021;42(2):101. doi:10.1210 / ENDREV / BNAA032 38. Leung AKK, Tam YYC, Chen S, Hafez IM, Cullis PR. Microfluidic Mixing: A General Method for Encapsulating Macromolecules in Lipid Nanoparticle Systems. J Phys Chem B. 2015;119(28):8698-8706. doi:10.1021 / ACS.JPCB.5B02891 / ASSET / IMAGES / LARGE / JP-2015- 028912_0009.JPEG 39. M. Hammer M, S. Wehrman T, M. Blau H. A novel enzyme complementation-based assay for monitoring G-protein-coupled receptor internalization. FASEB J.2007;21(14):3827- 3834. doi:10.1096 / fj.07-8777com 40. Zhao X, Jones A, Olson KR, et al. A Homogeneous Enzyme Fragment Complementation-Based -Arrestin Translocation Assay for High-Throughput Screening of G- Protein-Coupled Receptors. J Biomol Screen.2008;13(8):737-747. doi:10.1177 / 1087057108321531 41. Engsbli J, Kleinmans WA, Singhll L, Singhi G, Raufmanll JP. Isolation and Characterization of Exendin-4, an Exendin-3 Analogue, from Heloderma suspecturn Venom FURTHER EVIDENCE FOR AN EXENDIN RECEPTOR ON DISPERSED ACINI FROMMBHB Ref. No.24-0108-WO GUINEA PIG PANCREAS*. J Biol Chem. 1992;267(11):7402-7405. doi:10.1016 / S0021- 9258(18)42531-8 42. Rajabi H, Ahmadi M, Aslani S, Saberianpour S, Rahbarghazi R. Exendin-4 as a Versatile Therapeutic Agent for the Amelioration of Diabetic Changes. Adv Pharm Bull. 2022;12(2):237. doi:10.34172 / APB.2022.025 43. Fisher MM, Perez Chumbiauca CN, Mather KJ, Mirmira RG, Tersey SA. Detection of Islet -Cell Death in Vivo by Multiplex PCR Analysis of Differentially Methylated DNA. Endocrinology.2013;154(9):3476-3481. doi:10.1210 / en.2013-1223 44. Anderson MS, Bluestone JA. THE NOD MOUSE: A Model of Immune Dysregulation. Annu Rev Immunol.2005;23(1):447-485. doi:10.1146 / annurev.immunol.23.021704.115643 45. Hrovatin K, Bastidas-Ponce A, Bakhti M, et al. Delineating mouse -cell identity during lifetime and in diabetes with a single cell atlas. Nat Metab 202359.2023;5(9):1615-1637. doi:10.1038 / s42255-023-00876-x 46. Han Y, Liu D, Li L. PD-1 / PD-L1 pathway: current researches in cancer. Am J Cancer Res.2020;10(3):727. 47. Falcone M, Fousteri G. Role of the PD-1 / PD-L1 Dyad in the Maintenance of Pancreatic Immune Tolerance for Prevention of Type 1 Diabetes. Front Endocrinol.2020;11:569. doi:10.3389 / FENDO.2020.0056948. Wang L, Qi C, Cao H, et al. Engineered Cytokine Primed Extracellular Vesicles withHigh PD L1 Expression Ameliorate Type 1 Diabetes. Small. 2023;19(38):2301019.doi:10.1002 / smll.202301019 49. Muralidharan C, Huang F, Enriquez JR, et al. Inhibition of the eukaryotic initiationfactor-2- kinase PERK decreases risk of autoimmune diabetes in mice. J Clin Invest. Publishedonline June 2024. doi:10.1172 / JCI176136 50. Parhiz H, Shuvaev VV, Pardi N, et al. PECAM-1 directed re-targeting of exogenous mRNA providing two orders of magnitude enhancement of vascular delivery and expression in lungs independent of apolipoprotein E-mediated uptake. J Control Release Off J Control Release Soc.2018;291:106-115. doi:10.1016 / j.jconrel.2018.10.015 51. Böttger R, Pauli G, Chao PH, AL Fayez N, Hohenwarter L, Li SD. Lipid-based nanoparticle technologies for liver targeting. Adv Drug Deliv Rev.2020;154-155:79-101. doi:10.1016 / j.addr.2020.06.017MBHB Ref. No.24-0108-WO 52. Tombácz I, Laczkó D, Shahnawaz H, et al. Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Mol Ther. 2021;29(11):3293-3304. doi:10.1016 / j.ymthe.2021.06.004 53. Kularatne RN, Crist RM, Stern ST. The Future of Tissue-Targeted Lipid Nanoparticle- Mediated Nucleic Acid Delivery. Pharmaceuticals. 2022;15(7):897. doi:10.3390 / ph15070897 54. Qiu M, Tang Y, Chen J, et al. Lung-selective mRNA delivery of synthetic lipid nanoparticles for the treatment of pulmonary lymphangioleiomyomatosis. Proc Natl Acad Sci. 2022;119(8):e2116271119. doi:10.1073 / pnas.2116271119 55. Hadjiyanni I, Baggio LL, Poussier P, Drucker DJ. Exendin-4 Modulates Diabetes Onset in Nonobese Diabetic Mice. Endocrinology. 2007;149(3):1338. doi:10.1210 / en.2007-1137 56. Flamez D, Roland I, Berton A, et al. A genomic-based approach identifies FXYD domaincontaining ion transport regulator 2 (FXYD2) a as a pancreatic beta cell-specific biomarker.Diabetologia.2010;53(7):1372-1383. doi:10.1007 / S00125-010-1714-Z / TABLES / 1 57. Balhuizen A, Massa S, Mathijs I, et al. A nanobody-based tracer targeting DPP6 for non- invasive imaging of human pancreatic endocrine cells. Sci Rep.2017;7:15130. doi:10.1038 / s41598-017-15417-2 58. Saunders DC, Brissova M, Phillips N, et al. Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody Targets Adult Human Pancreatic Cells for In Vitro and In Vivo Analysis. Cell Metab.2019;29(3):745-754.e4. doi:10.1016 / j.cmet.2018.10.007 59. El Khatib M, Sakuma T, Tonne JM, et al. -cell-targeted blockage of PD1 and CTLA4 pathways prevents development of autoimmune diabetes and acute allogeneic islets rejection. Gene Ther. 2015;22(5):430-438. doi:10.1038 / gt.2015.18 60. dos Santos Haber JF, Barbalho SM, Sgarbi JA, et al. The Relationship between Type 1Diabetes Mellitus, TNF- , and IL-10 Gene Expression. Biomedicines. 2023;11(4):1120.doi:10.3390 / biomedicines11041120 61. Perez-Serna AA, Dos Santos RS, Ripoll C, Nadal A, Eizirik DL, Marroqui L. BCL-XL Overexpression Protects Pancreatic -Cells against Cytokine- and Palmitate-Induced Apoptosis. Int J Mol Sci.2023;24(6):5657. doi:10.3390 / ijms24065657 62. Fife BT, Guleria I, Bupp MG, et al. Insulin-induced remission in new-onset NOD mice is maintained by the PD-1–PD-L1 pathway. J Exp Med.2006;203(12):2737. doi:10.1084 / jem.20061577MBHB Ref. No.24-0108-WO 63. Yoshihara E, O’Connor C, Gasser E, et al. Immune-evasive human islet-like organoids ameliorate diabetes. Nat 20205867830. 2020;586(7830):606-611. doi:10.1038 / s41586-020- 2631-z 64. Bluestone JA, Buckner JH, Herold KC. Immunotherapy: Building a bridge to a cure for type 1 diabetes. Science.2021;373(6554):510-516. doi:10.1126 / SCIENCE.ABH1654 / ASSET / D6A9B56C-2521-48FE-A0AD- A979F076BD93 / ASSETS / IMAGES / LARGE / SCIENCE.ABH1654-F2.JPG 65. Wortham M, Sander M. Transcriptional mechanisms of pancreatic -cell maturation and functional adaptation. Trends Endocrinol Metab TEM. 2021;32(7):474. doi:10.1016 / J.TEM.2021.04.011 66. Sims EK, Bahnson HT, Nyalwidhe J, Haataja L, Davis AK, Speake C, DiMeglio LA, Blum J, Morris MA, Mirmira RG, Nadler J, Mastracci TL, Marcovina S, Qian WJ, Yi L, Swensen AC, Yip-Schneider M, Schmidt CM, Considine RV, Arvan P, Greenbaum CJ, Evans- Molina C, T1D Exchange Residual C-peptide Study Group. Proinsulin Secretion Is a Persistent Feature of Type 1 Diabetes. Diabetes Care.2019;42(2):258–64. 67. Sims EK, Chaudhry Z, Watkins R, Syed F, Blum J, Ouyang F, Perkins SM, Mirmira RG, Sosenko J, DiMeglio LA, Evans-Molina C. Elevations in the Fasting Serum Proinsulin-to-C- Peptide Ratio Precede the Onset of Type 1 Diabetes. Diabetes Care.2016 Sep;39(9):1519–26. 68. Maganti A, Evans-Molina C, Mirmira R. From immunobiology to -cell biology: the changing perspective on type 1 diabetes. Islets.2014;6(2):e28778. 69. Tersey SA, Nishiki Y, Templin AT, Cabrera SM, Stull ND, Colvin SC, Evans-Molina C, Rickus JL, Maier B, Mirmira RG. Islet -cell endoplasmic reticulum stress precedes the onset of type 1 diabetes in the nonobese diabetic mouse model. Diabetes.2012 Apr;61(4):818–27. 70. Huang RT, Wu D, Meliton A, Oh MJ, Krause M, Lloyd JA, Nigdelioglu R, Hamanaka RB, Jain MK, Birukova A, Kress JP, Birukov KG, Mutlu GM, Fang Y. Experimental Lung Injury Reduces Krüppel-like Factor 2 to Increase Endothelial Permeability via Regulation of RAPGEF3-Rac1 Signaling. Am J Respir Crit Care Med.2017 Mar 1;195(5):639–51. 71. Kuo CH, Leon L, Chung EJ, Huang RT, Sontag TJ, Reardon CA, Getz GS, Tirrell M, Fang Y. Inhibition of atherosclerosis-promoting microRNAs via targeted polyelectrolyte complex micelles. J Mater Chem B.2014 Dec 14;2(46):8142–53.MBHB Ref. No.24-0108-WO 72. Yeh CF, Cheng SH, Lin YS, Shentu TP, Huang RT, Zhu J, Chen YT, Kumar S, Lin MS, Kao HL, Huang PH, Roselló-Sastre E, Garcia F, Jo H, Fang Y, Yang KC. Targeting mechanosensitive endothelial TXNDC5 to stabilize eNOS and reduce atherosclerosis in vivo. Sci Adv. 2022 Jan 21;8(3):eabl8096. 73. Pugliese A. Insulitis in the pathogenesis of type 1 diabetes: Insulitis in type 1 diabetes. Pediatric Diabetes.2016 Jul;17:31–6. 74. Ovalle F, Grimes T, Xu G, Patel AJ, Grayson TB, Thielen LA, Li P, Shalev A. Verapamil and beta cell function in adults with recent-onset type 1 diabetes. Nature Medicine.2018 Aug;24(8):1108–12. 75. Hernandez-Perez M, Chopra G, Fine J, Conteh AM, Anderson RM, Linnemann AK, Benjamin C, Nelson JB, Benninger KS, Nadler JL, Maloney DJ, Tersey SA, Mirmira RG. Inhibition of 12 / 15-Lipoxygenase Protects Against -Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes. Diabetes.2017 Nov;66(11):2875–87. 76. Kulkarni A, Pineros AR, Walsh MA, Casimiro I, Ibrahim S, Hernandez-Perez M, Orr KS, Glenn L, Nadler JL, Morris MA, Tersey SA, Mirmira RG, Anderson RM.12-Lipoxygenase governs the innate immune pathogenesis of islet inflammation and autoimmune diabetes. JCI Insight.2021 Jul 22;6(14):147812. 77. Tersey SA, Colvin SC, Maier B, Mirmira RG. Protective effects of polyamine depletion in mouse models of type 1 diabetes: implications for therapy. Amino Acids.2014 Mar;46(3):633–42. 78. Kim J, Hu C, Moufawad El Achkar C, Black LE, Douville J, Larson A, Pendergast MK, Goldkind SF, Lee EA, Kuniholm A, Soucy A, Vaze J, Belur NR, Fredriksen K, Stojkovska I, Tsytsykova A, Armant M, DiDonato RL, Choi J, Cornelissen L, Pereira LM, Augustine EF, Genetti CA, Dies K, Barton B, Williams L, Goodlett BD, Riley BL, Pasternak A, Berry ER, Pflock KA, Chu S, Reed C, Tyndall K, Agrawal PB, Beggs AH, Grant PE, Urion DK, Snyder RO, Waisbren SE, Poduri A, Park PJ, Patterson A, Biffi A, Mazzulli JR, Bodamer O, Berde CB, Yu TW. Patient-Customized Oligonucleotide Therapy for a Rare Genetic Disease. New England Journal of Medicine. 2019 Oct 24;381(17):1644–52. 79. Rinaldi C, Wood MJA. Antisense oligonucleotides: the next frontier for treatment of neurological disorders. Nat Rev Neurol. 2018 Jan;14(1):9–21.MBHB Ref. No.24-0108-WO 80. Wu D, Harrison DL, Szasz T, Yeh CF, Shentu TP, Meliton A, Huang RT, Zhou Z, Mutlu GM, Huang J, Fang Y. Single-cell metabolic imaging reveals a SLC2A3-dependent glycolytic burst in motile endothelial cells. Nat Metab.2021 May;3(5):714–27. 81. Colvin SC, Maier B, Morris DL, Tersey SA, Mirmira RG. Deoxyhypusine synthase promotes differentiation and proliferation of T helper type 1 (Th1) cells in autoimmune diabetes. J Biol Chem.2013 Dec 20;288(51):36226–35. 82. Kulkarni A, Anderson CM, Mirmira RG, Tersey SA. Role of Polyamines and Hypusine in Cells and Diabetes Pathogenesis. Metabolites.2022 Apr 12;12(4):344. 83. Wu C, Huang RT, Kuo CH, Kumar S, Kim CW, Lin YC, Chen YJ, Birukova A, Birukov KG, Dulin NO, Civelek M, Lusis AJ, Loyer X, Tedgui A, Dai G, Jo H, Fang Y. Mechanosensitive PPAP2B Regulates Endothelial Responses to Atherorelevant Hemodynamic Forces. Circ Res. 2015 Jul 31;117(4):e41–53. 84. Wu D, Huang RT, Hamanaka RB, Krause M, Oh MJ, Kuo CH, Nigdelioglu R, MelitonAY, Witt L, Dai G, Civelek M, Prabhakar NR, Fang Y, Mutlu GM. HIF-1 is required fordisturbed flow-induced metabolic reprogramming in human and porcine vascular endothelium. Elife.2017 May 30;6:e25217. 85. Nahrendorf M, Jaffer FA, Kelly KA, Sosnovik DE, Aikawa E, Libby P, Weissleder R. Noninvasive vascular cell adhesion molecule-1 imaging identifies inflammatory activation of cells in atherosclerosis. Circulation.2006 Oct 3;114(14):1504–11. 86. Pyke C, Heller RS, Kirk RK, Ørskov C, Reedtz-Runge S, Kaastrup P, Hvelplund A, Bardram L, Calatayud D, Knudsen LB. GLP-1 receptor localization in monkey and human tissue: novel distribution revealed with extensively validated monoclonal antibody. Endocrinology.2014 Apr;155(4):1280–90. 87. Richards P, Parker HE, Adriaenssens AE, Hodgson JM, Cork SC, Trapp S, Gribble FM, Reimann F. Identification and characterization of GLP-1 receptor-expressing cells using a new transgenic mouse model. Diabetes.2014 Apr;63(4):1224–33. 88. Roed SN, Wismann P, Underwood CR, Kulahin N, Iversen H, Cappelen KA, Schäffer L, Lehtonen J, Hecksher-Soerensen J, Secher A, Mathiesen JM, Bräuner-Osborne H, Whistler JL, Knudsen SM, Waldhoer M. Real-time trafficking and signaling of the glucagon-like peptide-1 receptor. Mol Cell Endocrinol. 2014 Feb 15;382(2):938–49.MBHB Ref. No.24-0108-WO 89. Syed F, Tersey SA, Turatsinze JV, Felton JL, Kang NJ, Nelson JB, Sims EK, Defrance M, Bizet M, Fuks F, Cnop M, Bugliani M, Marchetti P, Ziegler AG, Bonifacio E, Webb- Robertson BJ, Balamurugan AN, Evans-Molina C, Eizirik DL, Mather KJ, Arslanian S, Mirmira RG. Circulating unmethylated CHTOP and INS DNA fragments provide evidence of possible islet cell death in youth with obesity and diabetes. Clin Epigenetics. 2020 Jul 31;12(1):116. 90. Piñeros AR, Gao H, Wu W, Liu Y, Tersey SA, Mirmira RG. Single-Cell Transcriptional Profiling of Mouse Islets Following Short-Term Obesogenic Dietary Intervention. Metabolites. 2020 Dec 18;10(12). 91. Saw PE, Song EW. Phage display screening of therapeutic peptide for cancer targeting and therapy. Protein Cell.2019 Nov;10(11):787–807. 92. Oh E, Stull ND, Mirmira RG, Thurmond DC. Syntaxin 4 Up-Regulation Increases Efficiency of Insulin Release in Pancreatic Islets From Humans With and Without Type 2 Diabetes Mellitus. The Journal of Clinical Endocrinology & Metabolism. 2014 May;99(5):E866– 70.93. Babi A, Lamprianou S, Vinet L, Stransky-Heilkron N, Xayaphoummine C, Campo MA,Glombik H, Schulte A, Juretschke HP, Montet X, Meda P, Lange N. Multivalent glibenclamide to generate islet specific imaging probes. Biomaterials.2016 Jan;75:1–12. 94. Wang M, Zhang Z, Huo Q, Wang M, Sun Y, Liu H, Chang J, He B, Liang Y. Targeted Polymeric Nanoparticles Based on Mangiferin for Enhanced Protection of Pancreatic - Cells and Type 1 Diabetes Mellitus Efficacy. ACS Appl Mater Interfaces. 2022 Mar 9;14(9):11092–103. 95. Islam, M. A.; Xu, Y.; Tao, W.; Ubellacker, J. M.; Lim, M.; Aum, D.; Lee, G. Y.; Zhou, K.; Zope, H.; Yu, M., Restoration of tumour-growth suppression in vivo via systemic nanoparticle-mediated delivery of PTEN mRNA. Nat. Biomed. Eng.2018, 2 (11), 850-864. 96. Lin, Y.-X.; Wang, Y.; Ding, J.; Jiang, A.; Wang, J.; Yu, M.; Blake, S.; Liu, S.; Bieberich, C. J.; Farokhzad, O. C., Reactivation of the tumor suppressor PTEN by mRNA nanoparticles enhances antitumor immunity in preclinical models. Sci. Transl. Med. 2021, 13 (599), eaba9772. 97. Nanomedicine and the COVID-19 vaccines. Nature Nanotechnology 202015:12. 2020;15(12):963-963. doi:10.1038 / s41565-020-00820-0 98. Sheoran S, Arora S, Samsonraj R, Govindaiah P, vuree S. Lipid-based nanoparticles for treatment of cancer. Heliyon.2022;8(5). doi:10.1016 / J.HELIYON.2022.E09403MBHB Ref. No.24-0108-WO 99. Ohaegbulam KC, Assal A, Lazar-Molnar E, Yao Y, Zang X. Human cancer immunotherapy with antibodies to the PD-1 and PD-L1 pathway. Trends Mol Med. 2015;21(1):24. doi:10.1016 / J.MOLMED.2014.10.009 100. Yu X, Li W, Young KH, Li Y. Posttranslational Modifications in PD-L1 Turnover and Function: From Cradle to Grave. Biomedicines. 2021;9(11). doi:10.3390 / BIOMEDICINES9111702 101. Krishnamurthy B, Lacorcia M, Kay TWH, Thomas HE, Mannering SI. Monitoring immunomodulation strategies in type 1 diabetes. Front Immunol.2023;14:1206874. doi:10.3389 / FIMMU.2023.1206874 / BIBTEX 102. Reed JC, Herold KC. Thinking bedside at the bench: the NOD mouse model of T1DM. Nature Reviews Endocrinology 201511:5.2015;11(5):308-314. doi:10.1038 / nrendo.2014.236 103. Cabrera O, Berman DM, Kenyon NS, Ricordi C, Berggren PO, Caicedo A. The unique cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl Acad Sci U S A.2006;103(7):2334-2339. doi:10.1073 / PNAS.0510790103 / ASSET / 18282175- 0741-4EC3-A3B5-CC4D80C1D5BC / ASSETS / GRAPHIC / ZPQ0060611030005.JPEG 104. Zhou Z, Zhu J, Yeh CF, et al. Precision mRNA Nanomedicine for Targeted Vascular Therapies in ARDS and Atherosclerosis. Published online January 14, 2025:2025.01.09.632179. doi:10.1101 / 2025.01.09.632179. 105. Sachs S, Bastidas-Ponce A, Tritschler S, et al. Targeted pharmacological therapy restores -cell function for diabetes remission. Nat Metab 202022.2020;2(2):192-209. doi:10.1038 / s42255-020-0171-3. 106. Zhu X, Xu Y, Solis LM, et al. Long-circulating siRNA nanoparticles for validating Prohibitin1-targeted non-small cell lung cancer treatment. Proc Natl Acad Sci U S A. 2015;112(25):7779-7784. doi:10.1073 / PNAS.1505629112 / SUPPL_FILE / PNAS.1505629112.SAPP.PDF 107. Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG. Mouse islet of Langerhans isolation using a combination of purified collagenase and neutral protease. J Vis Exp JoVE. 2012;(67). doi:10.3791 / 4137. 108. Miyazaki JI, Araki K, Yamato E, et al. Establishment of a Pancreatic Cell Line That Retains Glucose-Inducible Insulin Secretion: Special Reference to Expression of Glucose Transporter Isoforms. Endocrinology.1990;127(1):126-132. doi:10.1210 / ENDO-127-1-126.MBHB Ref. No.24-0108-WO 109. Miyazaki S, Tashiro F, Tsuchiya T, Sasaki K, Miyazaki J ichi. Establishment of a long- term stable -cell line and its application to analyze the effect of Gcg expression on insulin secretion. Sci Rep.2021;11(1):477. doi:10.1038 / S41598-020-79992-7.110. Tsonkova VG, Sand FW, Wolf XA, et al. The EndoC- H1 cell line is a valid model ofhuman beta cells and applicable for screenings to identify novel drug target candidates. Mol Metab.2018;8:144. doi:10.1016 / J.MOLMET.2017.12.007.

Claims

MBHB Ref. No.24-0108-WO What is claimed is:

1. A targeted nanoparticle, comprising: a) a lipid; b) a -cell specific targeting peptide; and c) a nucleic acid molecule.

2. The targeted nanoparticle of claim 1, wherein the lipid comprises: a) a polyamidoamine (PAMAM) dendrimer (G0-C14); b) cholesterol; c) polyethylene glycol 2000 (PEG); d) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE- PEG); and e) dioleoylphospha-tidylethanolamine (DOPE).

3. The targeted nanoparticle of claim 2, wherein the lipid comprises a molar ratio of: a) the polyamidoamine (PAMAM) dendrimer (G0-Cl4) to DSPE-PEG of about 2:1 to about 15:l; b) cholesterol to DSPE-PEG of about 15:1 to about 40:1; and c) DOPE to DSPE-PEG of about 15:1 to about 30:

1.

4. The targeted nanoparticle of claim 2, wherein the DSPE-PEG comprises a PEG domain comprising PEG having an average molecular weight of about 1,000 to about 100,000 Daltons.

5. The targeted nanoparticle of any one of claims 1-4, wherein the lipid encapsulates the nucleic acid molecule.

6. The targeted nanoparticle of any one of claims 1-5, wherein the lipid encapsulates about 1 M to about 100 M of the nucleic acid molecule.

7. The targeted nanoparticle of claim 1, wherein the -cell specific targeting peptide is a glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3MBHB Ref. No.24-0108-WO (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), or an FXYD domain containing iontransport regulator 2 a (FXYD2 a).

8. The targeted nanoparticle of claim 7, wherein the -cell specific targeting peptide is a glucagon-like peptide-1 (GLP-1).

9. The targeted nanoparticle of claim 1, wherein the targeted nanoparticle comprises about 3×10-10μM to about 2×10-8μM of the -cell specific targeting peptide.

10. The targeted nanoparticle of claim 1, wherein the nucleic acid molecule comprises an RNA molecule encoding an immune-regulatory protein.

11. The targeted nanoparticle of claim 10, wherein the immune regulatory protein is an immune checkpoint protein, a cytokine, or -cell transcriptional factor.

12. The targeted nanoparticle of claim 10, wherein the RNA molecule is an mRNA molecule.

13. The targeted nanoparticle of claim 12, wherein the mRNA molecule is an immune- regulatory mRNA or -cell transcriptional factors.

14. The targeted nanoparticle of claim 10, wherein the immune checkpoint protein is PD-L1.

15. The targeted nanoparticle of any one of claims 1-14, wherein the targeted nanoparticle is configured to deliver the nucleic acid molecule to the pancreas.

16. A method of treating diabetes in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, and a nucleic acid encoding PD-L1 encapsulated within the lipid, wherein the targeted nanoparticle is preferentially targeted to a dysfunctional pancreatic cell,MBHB Ref. No.24-0108-WO thereby modifying diabetes disease progression, and preventing or reversing disease occurrence.

17. The method of claim 16, wherein the diabetes is Type 1 diabetes (T1D).

18. The method of claim 16, wherein the dysfunctional pancreatic cell is a -cell.

19. A method of treating, preventing, or delaying symptoms of pre-diabetes in a subject, comprising: administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, and a nucleic acid encoding PD-L1 encapsulated within the lipid, wherein the subject has stage 1 or stage 2 pre-diabetes, and wherein the targeted nanoparticle is preferentially targeted to a dysfunctional pancreatic cell; delivering the nucleic acid encoding PD-L1 to -cells, promoting -cell survival, restoring functional -cells, and reducing insulitis, thereby treating, preventing, or delaying symptoms of pre-diabetes in the subject.

20. A pharmaceutical composition, comprising: a) a therapeutically effective amount of targeted nanoparticles, each targeted nanoparticle comprising a lipid, a -cell specific targeting peptide, and a nucleic acid molecule encoding a therapeutic peptide encapsulated within the lipid; and b) a pharmaceutically acceptable carrier, solvent, adjuvant, and / or diluent.

21. The pharmaceutical composition of claim 18, wherein the lipid comprises: a) a polyamidoamine (PAMAM) dendrimer (G0-C14); b) cholesterol; c) polyethylene glycol 2000 (PEG); d) 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE- PEG); and e) dioleoylphospha-tidylethanolamine (DOPE).MBHB Ref. No.24-0108-WO The pharmaceutical composition of claim 21, wherein the lipid comprises a molar ratio of: a) the polyamidoamine (PAMAM) dendrimer (G0-Cl4) to DSPE-PEG of about 2:1 to about 15:l; b) cholesterol to DSPE-PEG of about 15:1 to about 40:1; and c) DOPE to DSPE-PEG of about 15:1 to about 30:

1.

23. The pharmaceutical composition of claim 21 or 22, wherein the DSPE-PEG comprises a PEG domain comprising PEG having an average molecular weight of about 1,000 to about 100,000 Daltons.

24. The pharmaceutical composition of any one of claims 20-23, wherein the -cell specific targeting peptide comprises a glucagon-like peptide-1 (GLP-1), an ectonucleoside triphosphate diphosphohydrolase-3 (NTPDase3), a dipeptidyl peptidase-like protein 6 (DPP6), and / or anFXYD domain containing ion transport regulator 2 a (FXYD2 a).

25. The pharmaceutical composition of any one of claims 20-24, wherein the therapeutic peptide comprises PD-L1, IL-12, IL-23, PAX6 and / or MAF-B.

26. The pharmaceutical composition of any one of claims 20-25, wherein the pharmaceutical composition is formulated for inhalation, insufflation, oral, intravenous, topical, ocular, buccal, systemic, nasal, injection, transdermal, rectal, or vaginal administration.

27. The pharmaceutical composition of claim 26, wherein the pharmaceutical composition is formulated for inhalation or intravenous administration.

28. A pharmaceutical composition for pancreatic delivery of an RNA molecule, comprising: a) a targeted nanoparticle comprising i) a lipid; ii) a -cell specific targeting peptide; andMBHB Ref. No.24-0108-WO iii) a nucleic acid molecule encoding a therapeutic peptide encapsulated within the lipid; and b) a pharmaceutically acceptable carrier, wherein the composition is formulated such that once administered to a subject, the targeted nanoparticle delivers the nucleic acid molecule to -cells in the pancreas.

Images