Compositions containing milk small extracellular vesicles and lipid nanoparticles, and methods for their use

Abstract

Methods and materials for effective delivery of nucleic acids (e.g., RNA molecules) to cells are provided herein. For example, methods and materials for using combinations of milk extracellular vesicles and lipid nanoparticles to deliver RNA therapies to cells are provided herein.

Description

[0001] COMPOSITIONS CONTAINING MILK SMALL EXTRACELLULAR VESICLES AND LIPID NANOPARTICLES, AND METHODS FOR THEIR USE

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority from U.S. Provisional Application No. 63 / 637,118, filed April 22, 2024. The disclosure of the prior application is considered part of (and is incorporated by reference in) the disclosure of this application.

[0004] STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with U.S. government support under HD098860 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.

[0006] TECHNICAL FIELD

[0007] This document relates to methods and materials for effective delivery of nucleic acids (e.g., RNA molecules) to cells. For example, this document relates to methods and materials for using combinations of milk extracellular vesicles and lipid nanoparticles to deliver RNA therapies to cells.

[0008] BACKGROUND

[0009] RNA therapies have emerged as a revolutionary approach with vast therapeutic potential, offering targeted interventions for a wide array of diseases. These therapies operate by harnessing cellular machinery to modulate gene expression, enabling precise regulation of protein production or modification of genetic functions. However, RNA therapies require an appropriate delivery vehicle.

[0010] SUMMARY

[0011] This document is based, at least in part, on the development of methods and materials for efficient delivery of RNA therapies to cells (e.g., cells within a mammal). For example, this document is based, at least in part, on the discovery that the combinations of milk extracellular vesicles (EVs; referred to as “EXs” in U.S. Provisional Application No. 63 / 637,118) and lipid nanoparticles (LNPs) with ionizable lipids can serve as efficient delivery vehicles for RNA drugs. These hybrid delivery vehicles, referred to herein as “EVeLNPs” (referred to as “EXLs” in U.S. Provisional Application No. 63 / 637,118) can improve RNA translation and organ distribution. As described herein, EVeLNPs offer a promising avenue for treating genetic disorders, infectious diseases, cancer, and various other medical conditions, and provide a new method for delivery of personalized medicine and targeted interventions.

[0012] In a first aspect, this document features a composition containing, consisting of, or consisting essentially of: a milk extracellular vesicle (EV) and a lipid nanoparticle (LNP) comprising a nucleic acid cargo. The milk EV can be from human breast milk. The composition can contain the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein. The LNP can include an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound. The lipidoid can be an ionizable lipid including 3060iio, the cholesterol or derivative thereof can be cholesterol, the helper lipid can be l,2-dioleoyl-sn-glycero-3- phosphoethanolamine, and the PEG-based compound can be PEG-2000. The composition can contain the LNP in an amount of about 0.1 ng / mL mRNAto about 100 pg / mL mRNA. The composition can further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include one or more of water, saline, sucrose, dextrose, and trehalose. The nucleic acid can include one or more of an mRNA, siRNA, saRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer. The nucleic acid can include DNA.

[0013] In another aspect, this document features a method for delivering a nucleic acid to a cell. The method can include, consist of, or consist essentially contacting the cell with a composition containing: a milk extracellular vesicle (EV) and a lipid nanoparticle (LNP) containing a nucleic acid cargo. The milk EV can be from human breast milk. The composition can contain the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein. The LNP can include an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound. The lipidoid can be an ionizable lipid including 3060iio, the cholesterol or derivative thereof can be cholesterol, the helper lipid can be l,2-dioleoyl-sn-glycero-3- phosphoethanolamine, and the PEG-based compound can be PEG-2000. The composition can contain the LNP in an amount of about 0.1 ng / mL mRNAto about 100 pg / mL mRNA. The composition can further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include water, saline, sucrose, dextrose, or trehalose. The nucleic acid can include one or more of an mRNA, siRNA, saRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer. The nucleic acid can include DNA. The cell can be in vitro, and the method can include contacting the cell with the composition in an amount of about 10 ng to about 1 pg mRNA and / or about 0.5 pg / mL to about 100 pg / mL EV protein. The cell can be within a mammal (e.g., a human). The mammal can have been identified as having an infectious disease, autoimmune disease, cardiovascular disease, respiratory disease, neurological disease, cancer, metabolic disease, musculoskeletal disease, hematologic disease, or any combination thereof. The mammal can have been identified as having a condition or disease characterized by aberrant protein expression. The contacting can include contacting the cell with the composition in an amount of about 0.05 to about 5 mg / kg mRNA and / or about 0.1 to about 10 mg / kg EV protein.

[0014] In another aspect, this document features a method for delivering a messenger RNA to a cell within a mammal. The method can include, consist of, or consist essentially of administering, to the mammal, a composition containing: a milk extracellular vesicle (EV) and a lipid nanoparticle (LNP) containing a messenger RNA (mRNA) cargo. The milk EV can be from human breast milk. The composition can contain the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein. The LNP can include an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound. The lipidoid can be an ionizable lipid including 3060iio, the cholesterol or derivative thereof can be cholesterol, the helper lipid can be l,2-dioleoyl-sn-glycero-3 -phosphoethanolamine, and the PEG-based compound can be PEG-2000. The composition can contain the LNP in an amount of about 0.1 ng / mL mRNA to about 100 pg / mL mRNA. The composition can further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can include water, saline, sucrose, dextrose, or trehalose. The mammal can be a human. The administering can include administering the composition to the mammal in an amount of about 0.05 to about 5 mg / kg mRNA and / or about 0.1 to about 10 mg / kg EV protein. The cell can be a lung cell. The composition can be administered intravenously, intramuscularly, intraperitoneally, intrathecally, nasally, by inhalation, orally, or subcutaneously. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0015] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0016] DESCRIPTION OF DRAWINGS

[0017] FIGS.1A-1D illustrate characteristics of a hybrid delivery system formulated from LNPs and human EVs. To form a hybrid delivery system (EVeLNPs), small EVs obtained from human milk donors were gently mixed and incubated with mRNA-loaded LNPs (FIG. 1A). FIG. IB is a graph plotting total protein content of the EVs isolated from milk from for four human milk donors (designated A-D in the graph). The LNPs, EVs, and EVeLNPs were evaluated for diameter (n=8-17; FIG. 1C) and mRNA encapsulation efficiency (n=6-12; FIG. ID). Error bars represent s.e.m., with ns representing non-significance and **** representing p < 0.0001, according to one-way ANOVA with Dunnett’s multiple comparison test.

[0018] FIGS. 2A-2C show that EVeLNPs delivered mRNA more potently than lipid nanoparticles in vitro. LNPs encapsulating mLuc or EVeLNPs were incubated with HEK 293T epithelial cells (left panels) or RAW -Blue™ macrophages (right panels) for 24 hours at 100 ng / well of mLuc. FIG. 2A includes a pair of graphs plotting relative luminescence activity for LNPs or EVeLNPs that were formed by co-incubating LNPs and EVs for 0-20 hours at an EV protein concentration of 7.5 mg / mL, prior to their addition to cells (n=5). FIG. 2B includes a pair of graphs plotting relative luminescent intensity when EVeLNPs were incubated for 4 hours at increasing EV protein concentrations (indicated below the graph) prior to the addition of cells. Relative luminescent intensity of the LNP was measured for efficacy (n=10). FIG. 2C includes a pair of graphs plotting relative cell viability with EVeLNP treatment, determined using PBS as the negative control and TritonX (TrtX) as the positive control (n=5). Error bars represent s.e.m., **p<0.01, ***p<0.001, and 0.0001, according to one-way ANOVA with Dunnett' s multiple comparison test.

[0019] FIGS. 3A-3E show that EVeLNPs dramatically improved efficacy in the lungs. LNPs, EVs, or EVeLNPs were IV-injected into C57B / 6 mice at an mRNA dose of 0.1 mg / kg. After 3 hours, the organs were excised and imaged using the IVIS®. FIG. 3A is a graph plotting biodistribution, determined using LNPs encapsulating Cyanine5- (Cy5-) labeled mRNA. The corresponding EVeLNPs were generated using an EV protein concentration of 3.75 mg / kg (n=3). FIGS. 3B-3C show the results of efficacy experiments conducted with LNPs formulated with mLuc and the ionizable lipidoid 3060iio. EVeLNPs were generated from incubated with EVs at increasing EV protein concentration. Organs were imaged by IVIS® (FIG. 3B) and total luminescent flux was quantified (FIG. 3C), demonstrating increasing lung tropism with higher EV protein concentrations (n=4). FIGS. 3D-3E show the results of experiments performed as in FIGS. 3B-3C, but with several different ionizable lipids (MC3, SM-102, and ALC-0315, as indicated) and an EV protein concentration of 3.75 mg / kg (n=4). Error bars represent s.e.m. ****p < 0.0001 according to two-way ANOVA with Tukey’s multiple comparison test.

[0020] FIGS. 4A-4F show that EVeLNPs increased protein expression in lung cells. LNPs, EVs, or EVeLNPs were IV-injected into Ai9 mice. The LNPs encapsulated Cre Recombinase mRNA and were injected at a dose of 1 mg / kg mRNA. The EVs were injected at a dose of 3.75 mg / kg. Animals were sacrificed after 3 days, and their organs were harvested and imaged using the IVIS®. IVIS® images were obtained (FIG. 4A) and the total radiant efficiency was determined (FIG. 4B), showing an increase in lung translation (n=3). The liver and lung were then analyzed using flow cytometry. FIG. 4C is a graph plotting the percentage of tdTomato+ cells in the indicated cell types in the liver (MDSC, myeloid-derived suppressor cells). FIG. 4D is a graph plotting the percentage of tdTomato+ epithelial and endothelial cells in the indicated cell types in the lung. The lung cell types tested were determined to show a significant increase in protein expression. VECs, pulmonary vascular endothelial cells; BECs, bronchial epithelial cells; LECs, lymphatic endothelial cells; and AECs, alveolar epithelial cells type 1 (AEC1) and type 2 (AEC2). FIG. 4E is a graph plotting the percentage of tdTomato+ immune cells in the lung, which also demonstrated increased expression. FIG. 4F includes images of lung and liver tissue, which were assessed for toxicity. Scale bars represent 100 pm. Error bars represent s.e.m. *p<0.05, **p<0.01, and ****p < 0.0001, according to two-way ANOVA with Tukey’s multiple comparison test.

[0021] FIG. 5 includes a series of plots showing the concentration and size of EVs from four different donors, determined using Nanoparticle Tracking Analysis.

[0022] FIG. 6 includes a series of transmission electron microscopy images showing the shape and size of EVs from the four different donors.

[0023] FIG. 7 is an image of a representative Western blot confirming EV identity following isolation from human milk. EVs were positive for CD9 and TSG101, and negative for the endoplasmic reticulum protein, calnexin.

[0024] FIG. 8 is a graph plotting the diameter of LNP, EV, and EVeLNP particles (n=5- 12).

[0025] FIG. 9 is a graph plotting the concentration of LNP, EV, and EVeLNP particles (n=5-12).

[0026] FIG. 10 is a graph plotting the Zeta potential of LNP, EV, and EVeLNP particles that were diluted or concentrated. Error bars represent s.e.m. ns, non-significant; *p < 0.05, according to one-way ANOVA with Dunnett’s multiple comparison test (n=5).

[0027] FIG. 11 is a graph plotting biodistribution for labeled EVs. The corresponding EVeLNPs were generated using an EV protein concentration of 3.75 mg / kg and LNPs with the ionizable lipid 3060iio encapsulating mLuc (n=3).

[0028] FIG. 12 includes a series of pie charts showing expression distribution for LNPs and EVeLNPs that were IV-injected into C57B / 6 mice at an mRNA dose of 0.1 mg / kg and EV protein concentrations of 1.25, 3.75, or 5 mg / kg. After 3 hours, organs were excised and imaged using the IVIS®. Total luminescent flux was quantified and the percentage of protein expression occurred per organ is illustrated (n=4).

[0029] FIGS. 13A-13I are a series of graphs plotting levels of serum cytokines and chemokines, measured three hours, 24 hours, 48 hours, and 156 hours after IV-injection of EVs, LNPs, or EVeLNPs into C57B / 6 mice at an mRNA dose of 0.1 mg / kg. Cytokines and chemokines measured included CXCL1 (FIG. 13A), CXCL2 (FIG. 13B), interleukin- 1 alpha (IL- la, FIG. 13C), IL-6 (FIG. 13D), vascular endothelial growth factor (VEGF, FIG. 13E), IL-ip (FIG. 13F), tumor necrosis factor-alpha (TNF-a, FIG. 13G), CCL2 (FIG. 13H), and IL- 10 (FIG. 131). FIG. 14 is a graph plotting total luminescent flux for EVs that were isolated from mouse milk, bovine milk, goat milk, mouse macrophage cells, human serum, and human milk, and combined with 3060iio LNPs encapsulating mLuc to form EVeLNPs. These were IV-injected into C57BL / 6 mice at an mRNA dose of 0.1 mg / kg and an EV dose of 3.75 mg / kg. After 3 hours, the organs were excised and imaged using the IVIS®. The total luminescent flux was quantified, showing that only human EVeLNPs increased lung tropism. Error bars represent s.e.m. *p < 0.05, ***p < 0.001, and ****p < 0.0001, according to two-way ANOVA with Tukey’s multiple comparison test.

[0030] FIG. 15 is a graph plotting Cy5 signal for isotype control -EVeLNP (negative control), anti-CD9-EVeLNPs, anti-PEG-EVeLNPs, and anti-PEG-LNPs (positive control).

[0031] DETAILED DESCRIPTION

[0032] RNA therapeutics offer a broad range of applications targeted toward managing diseases characterized by disrupted protein expression (e.g., overexpression or underexpression), as well as for vaccination purposes. RNA-based drugs can directly address the genetic mechanisms underlying abnormal protein production, thereby presenting remedies for a diverse spectrum of medical conditions. Challenges in the application of mRNA-based therapeutics can include efficiency of mRNA delivery, mRNA stability, and immune response of a recipient against the therapeutics. Successful mRNA-based therapeutics typically rely on efficiently delivering mRNA molecules into target cells while avoiding degradation of the mRNAs by nucleases, enhancing mRNA stability to ensure they reach and remain functional at their targets, and minimizing the immune responses to exogenous mRNA, which can reduce or prevent inflammation and / or other adverse effects. The compositions and methods provided herein can achieve efficient delivery of mRNAs to cells in vitro and in vivo.

[0033] This document is based, at least in part, on the development of methods and materials for efficient delivery of nucleic acid (e.g., mRNA) therapies to cells (e.g., cells within a mammal). For example, this document provides materials and methods related to combinations of milk EVs and lipid-containing particles (e.g., LNPs containing ionizable lipids), and use of the combinations (referred to herein as EVeLNPs) as efficient delivery vehicles for nucleic acids (e.g., RNA drugs). Milk EVs can be used as delivery vehicles for exogenous drugs, but they typically are difficult to load with RNA with high efficacy. LNPs with ionizable lipids can efficiently deliver RNA drugs, but the hybrid delivery vehicle provided herein can enhance the stability and bioavailability of RNA, can improve RNA translation, and can improve organ distribution of RNA cargo.

[0034] Compositions

[0035] Provided herein are compositions containing milk EVs (e.g., human milk EVs) and one or more lipid-containing particle (e.g., LNPs) for delivery of one or more therapeutic agents (e.g., a nucleic acid, such as an mRNA).

[0036] In general, EVs can contain bioactive lipids, membrane and cytosolic proteins (e.g., tetraspanins, lactadherin, and immunoglobulins), and nucleic acids such as microRNAs and mRNAs. The EVs in the compositions provided herein can be from any appropriate type of milk. In some cases, a composition provided herein can include human milk EVs. In some cases, a composition provided herein can include milk EVs from a non-human mammal (e.g., a mouse, rat, cow, sheep, goat, or pig). The EVs can be prepared using any appropriate method. In some cases, EVs can be prepared by subjecting milk to ultracentrifugation and size exclusion chromatography. In some cases, EVs can be prepared using density gradient centrifugation or ultrafiltration, casein micelle removal, and immunoaffmity capture. See, e.g., Di et al., Front. Nutr., vol. 11, 2024; doi.org / 10.3389 / fnut.2024.1512939.

[0037] Lipid-containing particles are small particles or structures that include lipids and / or lipid-like materials (e.g., lipidoids). Lipids include, without limitation, fats, waxes, sterols, fat-soluble vitamins, and other similar substances. Lipidoids are a class of lipid- like materials often used in biotechnology and nanomedicine, particularly for the delivery of nucleic acids, such as RNA and DNA. Lipid-containing particles can be found in various biological forms such as LNPs, lipoproteins (which transport lipids in the bloodstream), lipid droplets (intracellular storage organelles), exosome (involved in cell- to-cell communication), vesicles (small membrane-bound sacs within cells), and others.

[0038] As described herein, lipid-containing particles (e.g., LNPs) can be effective for delivering nucleic acids and nucleic acid analogs to cells, often in vivo (see, e.g., Kulkarni et al., Nucleic Acid Therapeutics, 28(3): 146-157 , 2018; Hajj and Whitehead, Nature Reviews Materials, 2: 17056, 2017; U.S. Publication No. 20130245107; and U.S. Patent No. 8,754,062). In various embodiments, a lipid-containing particle (e.g., a LNP) can include a mixture of an ionizable and / or cationic lipid or lipidoid, cholesterol or a cholesterol analogue, a helper lipid such as DSPC, DOPC (dioleoyl phosphatidylcholine), DSPE (distearoyl phosphatidylethanolamine), or DOPE, and a polyethylene glycol-lipid conjugate (PEG-lipid) or PEG-cholesterol conjugate (PEG-cholesterol), as well as a polyanionic therapeutic agent such as a nucleic acid or nucleic acid analog. For targeting purposes, the PEG-lipid or PEG-cholesterol can be modified with a targeting moiety, such as N-acetylgalactosamine (GalNAc) for liver targeting, or with another ligand or binding reagent, such as an antibody or antibody fragment.

[0039] The lipid-containing particles (e.g., LNPs) in the compositions provided herein can include any appropriate lipid or lipidoid, cholesterol or cholesterol derivative, helper lipid, and polyethylene glycol (PEG)-based compound. Lipidoids are ionizable lipids, and non-limiting examples of suitable lipidoids include 3060iio, 2000iio, SM-102, DLin- MC3-DMA, ALC-0315, 3060io, 5030iio, 40206,IO, 500X1, 5000iio; 306On, 3060io, 306012, 200X6, 516iio, 50001, i, 8, 514X6, 306014, 501X1, 205016, 500013, 503O8,i2, 1130iio, 306016, 306013, 205018, 509X7, 5010iio, 5030iio, 500014, 1130iio, 509X1, 509X3, 501X2, 40206,IO, 51604,8, 402X8, 50101, 1,8, 50901, 1,8, and any combination thereof. In some embodiments, the lipidoid(s) or ionizable lipids in a LNP can include 3060iio, 2000iio, SM-102, DLin-MC3-DMA, ALC-0315, or any combination thereof. For example, the lipidoid or ionizable lipid contained in a LNP can be 3060iio, 2000iio, SM-102, DLin-MC3-DMA, or ALC-0315.

[0040] Helper lipids are cationic, anionic, neutral, or zwitterionic amphiphilic lipids that, along with cholesterol or a derivative thereof (e.g., a cholesterol analog), can aid in the molecular packing and stability of a lipid-containing particle (e.g., a LNP). Helper lipids also can enhance lipid nanoparticle efficacy by promoting fusion with both cell and endosomal membranes, facilitating cell uptake and endosomal release. Non-limiting examples of suitable helper lipids include DSPC, DSPE, DOPC, and DOPE. Additional useful helper lipids include phosphatidylcholine lipids. For example, a helper lipid can include l,2-distearoyl-sn-glycero-3-phosphocholine. Non-limiting examples of suitable cholesterol derivatives (or analogues) include oxidized cholesterol, desmosterol, 7- dehydrocholesterol, ergosterol, lanosterol, ketosterone, cholesterol sulfate, dehydroergosterol, cholestratrienol, 5-cholestene, and pregnenolone. In some cases, the lipid-containing particles (e.g., LNPs) in the compositions provided herein can contain cholesterol. PEG is a polyether compound derived from petroleum. PEG and PEG-based compounds can be used for various applications, such as drug delivery agents, solvents, adhesives, adsorbents, and tissue engineering scaffolds. PEG-based compounds include, without limitation, PEG-lipids (PEGylated lipids) and PEG-cholesterols (PEGylated cholesterols). PEG-lipids include a PEG moiety attached to one or more lipid moieties (e.g., a ceramide, succinoyl, or carbamate moiety). PEG-cholesterols include a PEG moiety attached to one or more cholesterol moieties. PEG-lipids and / or PEG-cholesterols can form a protective, non-aggregating, non-immunogenic shell around the surface of LNPs. Depending on the ultimate delivery route of the LNPs, the lipid group may be varied (e.g., in length) to dictate how long the PEG-lipid will be associated with the LNP, with longer lipid chains tending to remain associated with the LNP for longer time periods, and shorter lipid chains typically being useful for providing “diffusible” PEG lipids that diffuse from the lipid nanoparticle quickly to produce an LNP with increased transfection rates. The PEG moiety of a PEG-lipid or PEG-cholesterol can have a molecular weight ranging from about 300 g / mol to about 5000 g / mol (e.g., about 2000 g / mol, which is referred to as PEG 2000). Non-limiting examples of suitable PEG-lipids include 1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N [methoxy(polyethylene glycol)-2000], N-octanoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)2000]}, N-palmitoyl-sphingosine-l-{succinyl[methoxy(poly ethylene glycol)5000]}, 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-3000] (ammonium salt), l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(poly ethylene glycol)- 1000] (ammonium salt), and PEG-cholesterol, such as cholesterol -(polyethylene glycol-600). In some cases, the lipid-containing particles (e.g., LNPs) in the compositions provided herein can contain a PEG-lipid.

[0041] Lipid-containing particles (e.g., LNPs) can be generated using any appropriate method. For example, an LNP preparation can be generated by combining an ionizable lipid or lipidoid, cholesterol or a cholesterol derivative, a helper lipid, and a PEG-based compound in any appropriate amounts or ratios to generate LNPs. In some cases, LNPs can be prepared by combining: about 30 mol% to about 70 mol% (e.g., about 30 to 40 mol%, about 35 to 45 mol%, about 40 to 50 mol%, about 45 to 55 mol%, about 50 to 60 mol%, about 55 to 65 mol%, about to 60 to 70 mol%, about 30 mol%, about 40 mol%, about 50 mol%, about 60 mol%, or about 70 mol%) of a lipid or lipidoid (e.g., 3060iio); about 2 mol% to about 25 mol% (e.g., about 2 to 10 mol%, about 5 to 15 mol%, about 10 to 20 mol%, about 5 mol%, about 10 mol%, or about 15 mol%) of a helper lipid (e.g., DOPE); about 20 to about 60 mol% (e.g., about 20 to 30 mol%, about 25 to 35 mol%, about 30 to 40 mol%, about 35 to 45 mol%, about 40 to 50 mol%, about 45 to 55 mol%, about 50 to 60 mol%, about 30 mol%, about 35 mol%, about 38.5 mol%, about 40 mol%, about 45 mol%, or about 50 mol%) cholesterol or a cholesterol derivative (e.g., cholesterol); and about 0.2 to about 3 mol% (e.g., about 0.2 to 1 mol%, about 0.5 to 1.5 mol%, about 1 to 2 mol%, about 1.5 to 2.5 mol%, about 2 to 3 mol%, about 0.5 mol%, about 1 mol%, about 1.5 mol%, about 2 mol% or about 2.5 mol%) of a PEG-based compound (e.g., PEG-2000).

[0042] In some cases, the lipid-containing particles (e.g., LNPs) in the compositions provided herein can include one or more (e.g., one, two, three, four, or more than four) nucleic acids (e.g., mRNA) as cargo. As used herein, “nucleic acid” includes any compound and / or substance that comprises a polymer of nucleotides. In some embodiments, polymers of nucleotides are referred to as “polynucleotides.” Exemplary nucleic acids include, without limitation, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2’- amino-LNA having a 2 ’-amino functionalization, and 2’-amino-a-LNA having a 2’- amino functionalization) or hybrids thereof. Naturally occurring nucleic acids generally have a deoxyribose sugar (e.g., found in deoxyribonucleic acid (DNA)) or a ribose sugar (e.g., found in ribonucleic acid (RNA)).

[0043] Nucleic acids and nucleic acid analogs comprise a backbone and a sequence of nucleobases. In the context of the present disclosure, the backbone monomer residues can be any suitable nucleic acid backbone monomer residues having a negative charge, such as a ribose or deoxyribose connected to another ribose or deoxyribose by a phosphodiester bond, or a backbone residue of a nucleic acid analog monomer. The backbone monomer includes both the structural “residue” component, such as the ribose in RNA, and any active groups that are modified in linking monomers together, such as the 5’ triphosphate and 3’ hydroxyl groups of a ribonucleotide, which are modified when polymerized into RNA to leave a negatively -charged phosphodiester linkage.

[0044] In some cases, a nucleic acid cargo packaged in a lipid-containing particle (e.g., a LNP) can be a therapeutic agent. As used herein, a “therapeutic agent” is any compound or composition that can be delivered to a mammal (e.g., a human) to achieve a desired effect, such as a beneficial treatment or a curative effect. Therapeutic agents include, without limitation, nucleic acids, nucleic acid analogs, proteins, polypeptides, small molecule drugs, antibiotics, antivirals, and cell-based therapies (e.g., CAR-T cell therapies). For example, therapeutic agents can be nucleic acids or nucleic acid analogs having a negatively-charged backbone, including but not limited to single-stranded DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, or modified versions of any of the preceding (e.g., versions that include one or more changes to the nucleotide components), or analogs of any of the preceding (e.g., synthetic molecules that mimic the structure and function of the original). With regard to overall structure and function of the nucleic acid or nucleic acid analog, the nucleic acid or nucleic acid analog may be, without limitation: mRNA (messenger RNA), siRNA (small interfering RNA), saRNA (self-amplifying RNA), miRNA (microRNA), gRNA (guide RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), tmRNA (transfer-messenger RNA), IncRNA (long noncoding RNA), circRNA (circular RNA), antisense RNA, ncRNA (non-coding RNA), telomerase RNA, piRNA (Piwi-interacting RNA), snRNA (small nuclear RNA), snoRNA (small nucleolar RNA), scaRNAs (small Cajal body RNA), Y RNA, eRNA (enhancer RNA), shRNA (small hairpin RNA), stRNA (small temporal RNA), DNA, chloroplast DNA, cDNA (complementary DNA), gDNA (genomic DNA), Hachimoji DNA, mitochondrial DNA, msDNA (multicopy single-stranded DNA), XNA (xeno nucleic acid), glycol nucleic acid, threose nucleic acid, hexose nucleic acid, LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino oligomer, antisense oligonucleotide, ribozyme, deoxyribozyme, aptamer, cloning vector, phagemid, plasmid, lambda phage, cosmid, fosmid, or artificial chromosome.

[0045] RNA therapeutics are a class of RNA-based treatments that target specific genes or genetic pathways with high specificity. The use of RNA therapeutics allow for transient expression or inhibition, which can reduce long-term side effects. RNA therapeutics utilize various forms of RNA to treat diseases such as, without limitation, infectious diseases, cancer, genetic disorders, cardiovascular diseases, and neurological diseases. Research has been ongoing since the 1990s, with significant success in cancer therapy in the early 2010s (see, e.g., Sahin et al., Nature Reviews Drug Discovery, 13:759-780, 2014). The RNA used in an RNA therapeutic can include, for example, mRNA, siRNA, short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, gRNA, long non-coding RNA, transfer RNA, ribosomal RNA, double-stranded RNA (dsRNA), and / or an RNA aptamer. In some cases, for example, an mRNA-based therapy can be used. mRNA-based therapies can trigger synthesis of proteins by delivering coding mRNA into cells, making such therapies particularly useful in vaccine development (see, e.g., DeFrancesco, Nature Biotechnology, 35: 193-197, 2017). The coding mRNA can be designed as a blueprint to generate a protein of interest (e.g., a reporter protein, a functional protein, or an antigen). In some embodiments, a protein of interest can be an antigen produced by a pathogen (e.g., a virus) or by a cancer cell. Such protein molecules can stimulate an adaptive immune response that teaches the body to identify and destroy the corresponding pathogen or cancer cells (see, e.g., Bae and Park, Advanced Drug Delivery Reviews, 158:4-16, 2020). mRNA vaccines (e.g., the Pfizer-BioNTech COVID- 19 vaccine and the Modema COVID-19 vaccine) were developed for use in combating the coronavirus disease during the COVID-19 pandemic (see, Noor, Current Clinical Microbiology Reports, 8(3): 178-185, 2021). As described in the Examples herein, an mRNA cargo can encode a reporter polypeptide such as firefly luciferase or Cy5. An mRNA encoding a desired protein (e.g., an mRNA encoding a reporter polypeptide) can be delivered into cells using lipid-containing particles (e.g., LNPs) to produce the polypeptide (e.g., luciferase) in vitro, through cell culture, and in vivo, such as in mouse models or in any other appropriate mammal (e.g., humans, non-human primates, rats, rabbits, cows, pigs, sheep, dogs, and / or cats). The mammal can be healthy or can have a clinical condition or disorder.

[0046] In some cases, a LNP can include mRNA as a cargo (e.g., for in vitro use) in an amount from about 0.001 mg / mL to about 2 mg / mL (e.g., from about 0.001 mg / mL to about 1.5 mg / mL, from about 0.003 mg / mL to about 1.5 mg / mL, from about 0.005 mg / mL to about 1.5 mg / mL, from about 0.003 mg / mL to about 1 mg / mL, from about 0.003 mg / mL to about 0.5 mg / mL, from about 0.005 to about 1.5 mg / mL, from about 0.005 to about 1 mg / mL, from about 0.005 to about 0.5 mg / mL, about 0.001 mg / mL, about 0.003 mg / mL, about 0.005 mg / mL, about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.08 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 1.5 mg / mL, or about 2 mg / mL). In some cases, the concentration of mRNA cargo in an LNP (e.g., for in vivo use) can be from about 0.01 mg / mL to about 10 mg / mL (e.g., from about 0.01 mg / mL to about 1 mg / mL, from about 0.03 mg / mL to about 3 mg / mL, from about 0.05 mg / mL to about 5 mg / mL, from about 0.1 mg / mL to about 2 mg / mL, from about 0.3 mg / mL to about 3 mg / mL, from about 0.5 mg / mL to about 5 mg / mL, from about 1 mg / mL to about 3 mg / mL, from about 3 to about 5 mg / mL, about 0.01 mg / mL, about 0.03 mg / mL, about 0.05 mg / mL, about 0.1 mg / mL, about 0.3 mg / mL, about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, or about 10 mg / mL).

[0047] In some cases, the ratio of lipidoid:mRNA (weight / weight) in a LNP can be from about 5 to about 50 (e.g., about 5 to about 40, 5 to about 30, about 5 to about 20 about, 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 5, about 10, about 12.5, about 15, about 17.5, about 20, about 25, about 30, about 40, or about 50).

[0048] In some cases, the compositions provided herein can contain EVs and lipid- containing particles (e.g., LNPs packaged with one or more nucleic acids), in combination with one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition provided herein include, without limitation, water, sucrose, dextrose, trehalose, salts, or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), and buffers (e.g., phosphate buffered saline).

[0049] Methods

[0050] This document also provides methods for making and using EVeLNPs. EVeLNPs can be prepared using any appropriate method. In some cases, EVeLNPs can be generated by mixing EVs and LNPs (e.g., using gentle pipetting) and incubating the mixture at an appropriate temperature (e.g., about -2°C to about 0°C, about 0°C to about 3°C, about 3°C to about 10°C, about 3°C, about 4°C, about 5°C, or about 7°C) for an appropriate length of time (e.g., about 30 minutes to about 24 hours, about 1 to 2 hours, about 3 to 5 hours, about 4 to 6 hours, about 6 to 12 hours, or about 12 to 24 hours). The compositions provided herein can contain any appropriate amount of milk EVs (e.g., human milk EVs). In some cases, the amount of milk EVs included in an EVeLNP composition can be quantified based on the protein content of the EVs. For example, a composition provided herein can contain from about 0.1 mg / mL to about 15 mg / mL (e.g., about 0.1 to about 1 mg / mL, about 1 to about 3 mg / mL, about 3 to about 5 mg / mL, about 5 to about 7.5 mg / mL, about 7.5 to about 10 mg / mL, about 10 to about 15 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 5 mg / mL, about 7.5 mg / mL, or about 10 mg / ml) milk EVs, based on protein content. The protein content of an EV preparation can be determined using any appropriate assay or technique. For example, the protein content of an EV preparation can be determined using a bicinchoninic acid (BCA) assay.

[0051] The compositions provided herein can contain any appropriate amount of lipid- containing particles (e.g., LNPs). In some cases, the amount of nucleic acid-containing LNPs included in an EVeLNP composition provided herein can be quantified based on the nucleic acid (e.g., mRNA) content of the LNPs. For example, a composition provided herein can contain from about 0.1 ng / mL to about 100 pg / mL (e.g., about 0.1 to about 1 ng / mL, about 1 to about 10 ng / mL, about 10 to about 100 ng / mL, about 100 to about 500 ng / mL, about 500 ng / mL to about 1 pg / mL, about 1 to about 10 pg / mL, or about 10 to about 100 pg / mL) LNPs, based on nucleic acid (e.g., mRNA) content. The nucleic acid content of a LNP preparation can be determined using any appropriate assay or technique. For example, the nucleic acid content of a LNP preparation can be determined using a commercially available kit (e.g., the Quant-it™ RiboGreen Reagent available from ThermoFisher Scientific).

[0052] This document also provides methods for delivering a therapeutic agent (e.g., an mRNA) to a cell. The methods can include administering, to a cell, an EVeLNP composition provided herein, where the EVeLNP includes a lipid-containing particle (e.g., LNP) that encapsulates the therapeutic agent (e.g., mRNA). Methods that include delivering RNA into cells can be useful in research and therapeutic applications, including gene silencing, gene editing, and mRNA-based therapeutics. As described herein, RNA delivery can be achieved the EVeLNPs provided herein, which can avoid issues encountered with delivery of naked, single-stranded RNA (which is prone to nuclease degradation, can activate the immune system, and is too large and negatively charged to passively cross the cell membrane). The cell can be in vitro (e.g., a cell in culture) or in vivo (e.g., a cell in a subject such as a mammal). The methods provided herein can include administering any appropriate amount of an EVeLNP composition. When the cell is in vitro, for example, a method provided herein can include contacting the cell with an EVeLNP composition at an amount of about 10 ng to about 1 pg of mRNA (e.g., about 10 ng to about 50 ng, about 50 ng to about 100 ng, about 100 ng to about 250 ng, about 250 ng to about 500 ng, or about 500 ng to about 1 pg), and / or a final protein concentration of EVs at about 0.5 pg / mL to about 100 pg / mL (e.g., about 0.5 pg / mL to about 1 pg / mL, about 1 pg / mL to about 10 pg / mL, about 10 pg / mL to about 25 pg / mL, about 25 pg / mL to about 50 pg / mL, or about 50 pg / mL to about 100 pg / mL).

[0053] This document also provides methods that can be used to deliver a therapeutic agent to a subject (e.g., a mammal such as a human). Any appropriate subject can be treated as described herein. For example, humans or other primates such as monkeys can be treated by administering a composition containing EVeLNPs as described herein. In some cases, dogs, cats, horses, cows, pigs, sheep, mice, and / or rats can be treated by administering an EVeLNP composition as described herein. In some cases, the methods described herein can be used to deliver a therapeutic agent (e.g., a therapeutic nucleic acid such as an mRNA) to the lung of a subject (e.g., a mammal). The methods can include administering to the subject an EVeLNP composition containing EVs and a lipid- containing particle (e.g., a LNP) that includes the therapeutic agent. An EVeLNP composition can be administered to a subject by any appropriate route. For example, EVeLNPs can be administered to a subject (e.g., a mammal) intravenously, intramuscularly, intraperitoneally, intrathecally, nasally, by inhalation, orally, or subcutaneously.

[0054] EVeLNPs can be administered in any appropriate amount. In some cases, an EVeLNP composition containing EVs and LNP -packaged mRNA can be administered at an mRNA dose of about 0.05 mg / kg to about 5 mg / kg and / or an EV protein dose of about 0.1 to about 10 mg / kg. For example, an EVeLNP composition containing EVs and LNP- packaged mRNA can be administered at an mRNA dose of about 0.05 to about 5 mg / kg (e.g., about 0.05 to about 0.5 mg / kg, about 0.5 to about 1 mg / kg, about 1 to about 2 mg / kg, about 2 to about 3 mg / kg, about 3 to about 4 mg / kg, about 4 to about 5 mg / kg, or about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 2, about 3, about 4, or about 5 mg / kg), and / or at an EV protein dose of about 0.5 to about 10 mg / kg (e.g., about 0.1 to about 0.5 mg / kg, about 0.5 to about 1 mg / kg, about 1 to about 3 mg / kg, about 3 to about 5 mg / kg, about 5 to about 7 mg / kg, about 7 to about 10 mg / kg, or about 0.1, about 0.25, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 mg / kg). In some cases, an EVeLNP composition containing EVs and LNP -packaged mRNA can be administered at an mRNA dose of about 0.1 mg / kg and an EV protein dose of about 3 mg / kg.

[0055] As described herein, the therapeutic agent contained within the lipid-containing particle (e.g., LNP) used in the methods provided herein can be a nucleic acid (e.g., an RNA). The RNA as the therapeutic agent can be an mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamer. In some cases, an RNA encoding a marker (e.g., an mRNA encoding a luciferase polypeptide) can be used in the methods described herein. Luciferase is an enzyme that catalyzes a bioluminescent reaction, producing light as a byproduct. This reaction can be utilized in various biological and medical research applications, particularly in reporter assays to study gene expression and cellular processes. The bioluminescence produced by luciferase encoded by the mRNA encapsulated in the lipid-containing particle (e.g., LNP) can be used to assess efficacy of mRNA delivery.

[0056] In some cases, the methods provided herein can be used to treat a clinical disorder in a subject (e.g., a mammal). As used in this context, to “treat” means to reduce ameliorate at least one symptom of a disorder. For example, a treatment for an inflammatory condition in a mammal can result in a reduction in NF-KB levels in the mammal. Administration of a therapeutically effective amount of an EVeLNP composition as described herein can result in improved LNP efficacy of mRNA delivery. Thus, the methods described herein provide an approach to enhance the effectiveness of RNA therapeutics.

[0057] Disorders that can be treated according to methods provided herein include, without limitation, disorders and diseases in which one or more genes or proteins is dysregulated (e.g., such that a mammal with the disorder or disease has aberrant protein expression). In some cases, a disorder that can be treated as described herein can be associated with inflammation in a lung, liver, spleen, blood vessel, brain, lymph node, or gastrointestinal tract of a subject (e.g., a mammal). The inflammatory condition can be induced by a stimulator such as LPS or lipid-containing particles (e.g., LNPs), or can be due to an infectious disease, injury, toxin, chronic disease (e.g., cancer), smoking, or any combination thereof. In some cases, the disorder can be an infectious disease, an autoimmune disease, a cardiovascular disease, a respiratory disease, a neurodegenerative disease, a cancer, a genetic disorder a metabolic disease, a musculoskeletal disease, a hematologic disease, or any combination thereof.

[0058] Generally, the methods provided herein include administering a therapeutically effective amount of EVeLNPs that include EVs and lipid-containing particles (e.g., LNPs) as described herein, to a subject that is in need thereof or has been determined to be in need of, such treatment. The lipid-containing particles (e.g., LNPs) in the EVeLNPs can encapsulate a therapeutic agent for the treatment needed. The therapeutic agent can be mRNA, siRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, or RNA aptamers.

[0059] Effective doses can vary depending on the severity of the disorder, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments, and the judgment of the treating clinician. An effective amount of an EVeLNP composition containing EVs and lipid- containing particles (e.g., LNPs) as described herein can be any amount that reduces one or more symptoms of the disorder (e.g., by at least 10, 25, 35, 45, 50, 55, 65, 75, 80, 90, or 100 percent) within a subject (e.g., a mammal), without producing severe toxicity in the mammal. As described herein, for example, an effective dose of an EVeLNP can be an mRNA dose of mRNA dose of about 0.05 mg / kg to about 5 mg / kg and / or an EV protein dose of about 0.1 to about 10 mg / kg. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, severity of disorder, or risk level for development of the same or another disorder in the mammal being treated may require an increase or decrease in the actual effective amount administered.

[0060] If a particular mammal fails to respond to a particular amount of an EVeLNP composition, then the amount of the composition administered can be increased by, for example, two-fold. After receiving the higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments can be made accordingly. The frequency of administration of an EVeLNP composition containing EVs and lipid-containing particles (e.g., LNPs) provided herein to a subject (e.g., a mammal) can be any frequency that reduces a symptom of a disorder in the subject, without producing significant toxicity to the subject. For example, the frequency of administration of an EVeLNP composition can be from about four times daily to about once a day, from about once daily to three times a week, from about three times a week to about twice a week, from about twice a week to about once a week, from about once a week to about once every other week, from about once a week to about once a month, from about once a month to once a year, from about twice a year to about once a year, or from about once a year to once every several years). In some cases, an EVeLNP composition can be administered once (e.g., when the expression of the LNP cargo is a vaccine or encodes gene editing polypeptides that can edit a gene and permanently correct its expression). In some cases, a course of treatment with an EVeLNP composition described herein can include rest periods. For example, EVeLNPs can be administered daily over a one-week period followed by a one-week rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the disorder may require an increase or decrease in administration frequency.

[0061] An effective duration for administering an EVeLNP composition containing EVs and lipid-containing particles (e.g., LNPs) to a subject (e.g., a mammal) can be any duration that reduces a symptom of a disorder in the mammal, without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several days to several months. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the effective amount, frequency of administration, use of multiple treatments, route of administration, and severity of the disorder being treated.

[0062] In some cases, the progression of a disorder in a subject (e.g., a mammal) or the severity of one or more symptoms related to the disorder in the subject being treated can be monitored. Any appropriate method can be used to determine whether or not a subject having a disorder is effectively being treated. When the disorder is a cancer, for example, the progression of cancer in a subject (e.g., a mammal) or the severity of one or more symptoms related to the cancer in the subject being treated can be monitored. Any appropriate method can be used to determine whether or not a subject having cancer or at risk for developing cancer is effectively being treated. For example, clinical scanning techniques (e.g., computed tomography (CT), positron emission tomography (PET) / CT, bone scan, and magnetic resonance imaging (MRI)) can be used to determine the presence or absence of cancer within a mammal (e.g., a human) being treated. A reduced number of tumor cells and / or reduced tumor size can indicate effective treatment.

[0063] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0064] As used herein, the term “about,” when used herein in reference to a value, refers to a value that is ± 10% of the referenced value.

[0065] The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0066] EXAMPLES

[0067] Example 1 - EVeLNP formulation and characterization

[0068] Small EVs were isolated from human milk from four donors. LNPs were formulated using the ionizable lipid 3060iio in combination with DOPE, cholesterol, and C14-PEG-2000 at a molar ratio of 35: 16:46.5:2.5 by pipette mixing the lipid solution into the mRNA solutions being tested. The protein concentration of the EVs was quantified using the BCA assay. The average protein content was determined to be 1.1 mg / mL, with only slight variability of the content across the donors (FIG. IB). Nanoparticle Tracking Analysis (NTA) was used to determine the number of particles per mL and size of the EVs (FIG. 5). No significant difference was seen across donors. Transmission electron microscopy was used to evaluate the shape and size of EVs from the four different donors, and revealed no significant differences (FIG. 6). Western blotting was used to confirm EV identity; the EVs were positive for CD9 and TSG101 and negative for the endoplasmic reticulum protein, calnexin (FIG. 7).

[0069] The EVs and LNPs were then combined through gentle pipette mixing and incubation at 4°C to make EVeLNPs (FIG. 1A). The EVeLNPs were biophysically characterized in comparison to unmixed LNP and EV samples. Dynamic Light Scattering (DLS) and NTA were used to determine the size of the particles. Using DLS, their average diameters ranged from 100-115 nm, and while the EVeLNP particles were on average the largest, there was no statistically significant difference between the samples (FIG. 1C). Using NT A, the mean particle size of LNPs and EVs were not statistically different but there was an increase in size for the EVeLNPs (FIG. 8). The LNP-only and EV-only sizes were consistent with sizes disclosed elsewhere (Hu et al., Pharm. (Basel), 14(10): 1050, 2021; Hajj et al., Nano. Let., 20:5167-5175, 2020; and Thery et al., J. Extracell. Vesicles, 7: 1535750, 2018). The amount of firefly luciferase mRNA encapsulated by the mRNA+EVs, LNPs, and EVeLNPs was determined using the Quant- iT™ RiboGreen Assay (ThermoFisher Scientific). EVs mixed with mRNA did not encapsulate the mRNA, but 90% of the mRNA was encapsulated by the LNPs and the EVeLNPs (FIG. ID).

[0070] Concentration analysis of the number of particles demonstrated that EVs and EVeLNPs were less concentrated than LNPs (FIG. 9). In addition, analysis of the Zeta potential (surface charge) of LNP, EV, and EVeLNP particles demonstrated that the EVeLNPs were not statistically different than the LNPs, meaning that the uptake of EVeLNPs due to the charge of these particles should not be different than LNPs (FIG. 10).

[0071] Example 2 - EVeLNPs increased translation in vitro

[0072] The delivery of mRNA into cultured cells using EVs, LNPs, and EVeLNPs was assessed. First, the effect of incubation time for LNPs and EVs on delivery in cell culture was assessed. mRNA encoding firefly luciferase was encapsulated with LNPs. EVs were combined with the LNPs for two, four, six, or twenty hours at 4°C. The EVeLNPs were then added to HEK 293 T and RAW -Blue™ cells at a dose of 100 ng per well of mRNA, and a final protein concentration of EVs at 7.5 pg / mL. The HEK293 and RAW-Blue™ cell lines were selected for these studies because they are two different types of cells (epithelial and macrophages, respectively), and they are gold standards for testing mRNA transfection in vitro (see, e.g., Chaudhary et al., Proc. Natl. Acad. Sci., 121:e2307810121, 2024; and Pardi et al., J. Controlled Release, 217:345-351, 2015). After 24 hours, protein production was measured using the Bright-Glo™ assay (Promega Corp.). In both cell lines, protein expression was increased up to six orders of magnitude with the EVeLNPs, with EVeLNPs generated by incubating for 20 hours showing a decrease (FIG. 2A). An incubation time of 4 hours was used for subsequent experiments with EVeLNPs.

[0073] To determine how the concentration of EVs impacted delivery, the EVs were combined with LNPs for 4 hours and added to HEK 293T and RAW-Blue™ cells at 100 ng mRNA per well, with increasing EV protein concentrations of 0.5 to 20 pg / mL. After 24 hours, luciferase expression was measured using the Bright-Glo™ assay. In both cell types, the expression of luciferase was increased as EV protein concentration increased. For RAW -Blue™ cells, the EV protein concentration needed to be above 5 pg / mL to result in a significant increase in luciferase expression (FIG. 2B).

[0074] The MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for cell viability was used to assess the potential toxicity of the EVeLNP particles. EVeLNPs (after 4 hours of incubation) were added to HEK 293T and RAW-Blue™ cells at an mRNA dose of 100 ng per well with varying protein concentrations of EVs, and the cells were incubated for 24 hours. PBS used as the negative control, and Triton-X served as the positive control (lysing the cells). Cell viability did not substantially change with any treatment (FIG. 2C).

[0075] Example 3 - EVeLNPs increased mRNA translation in the lung in vivo

[0076] To build on the results showing that EVeLNPs increased protein production and did not cause toxicity in vitro, studies were conducted to determine whether EVeLNPs would increase or alter protein expression in vivo, and to determine whether the EVeLNPs had different organ tropism (biodistribution) compared to LNPs. LNPs encapsulating Cy5-tagged mRNA or mLuc were generated with 3060iio. The particles were intravenously injected into mice at a dose of 0.1 mg / kg mRNA and 3.75 mg / kg EV protein concentration, using fluorescently labeled EVs with the mLuc LNPs. Biodistribution was assessed among mouse organs after three hours, revealing that there was no change in biodistribution when EVs were included with the LNPs containing Cy5- tagged mRNA (FIG. 3A) or mLuc (FIG. 11).

[0077] To determine if protein expression was changed in vivo with the EVeLNPs, the EVeLNPs were incubated for four hours and then injected intravenously into mice at a dose of 0.1 mg / kg mRNA with EV protein concentration varying from 0.25 to 5 mg / kg. The LNP component of the EVeLNPs encapsulated firefly luciferase mRNA. A dosedependent increase in luciferase protein expression was observed with increasing EV protein concentration (FIGS. 3B-3C). The organ in which protein expression was increased the most was the lung, where overall protein expression increased by greater than two orders of magnitude in the EVeLNP group compared to LNP group (FIG. 3B). Total bioluminescence efficacy also was increased five-fold at the maximum EV protein concentration tested. Specifically, the fold increase for the lymph nodes, spleen, pancreas, kidney, and heart were 4-, 3-, 10-, 35-, and 23-fold, respectively. When normalized to total bioluminescence, efficacy did decrease in the liver by almost 2-fold in the EVeLNP group compared to the LNP group, although the total liver expression was still high (FIGS. 3B-3C). However, when further studies were conducted using LNPs or EVeLNPs that were IV-injected into C57B / 6 mice at an mRNA dose of 0.1 mg / kg and EV protein doses of 1.25, 3.75, or 5 mg / kg, total luminescent flux was progressively higher in the lung as the protein dose increased (FIG. 12).

[0078] Further studies were conducted to determine whether the increased protein expression observed in the lung with LNPs containing the ionizable lipid of 3060iio would also apply to other ionizable lipids. Three alternative ionizable lipids were tested: DLin-MC3-DMA, SM-102, and ALC-0315, all of which are FDA-approved LNP components. The EVeLNPs were intravenously injected into mice with firefly luciferase mRNA at 0.1 mg / kg and EVs at 3.75 mg / kg. For all ionizable lipids tested, there was a significant increase in protein expression in the lung (FIGS. 3D-3E). Specifically, the increase in protein expression observed in the lung with DLin-MC3-DMA, SM-102, and ALC-0315 was over three orders of magnitude compared to LNPs alone. In additional, the EVeLNPs contributed to an approximately 10-fold increase in total protein expression compared to LNPs alone (FIG. 3E).

[0079] Given the increased efficacy, studies were conducted to determine whether the shifts were due to an immune response. Blood levels of nine cytokines and chemokines were measured using Luminex beginning 3 hours post EVeLNP injection, revealing significant differences between most EV and EVeLNP groups and LNP and EVeLNP groups (FIGS 13A-13I). Studies also were conducted using EVeLNPs that included EVs isolated from mouse milk, bovine milk, goat milk, mouse macrophage cells, human serum, and human milk, and 3060iio LNPs encapsulating mLuc. These were IV-injected into C57BL / 6 mice at an mRNA dose of 0.1 mg / kg and an EV dose of 3.75 mg / kg. After 3 hours, the organs were excised and imaged using the IVIS®. The total luminescent flux was quantified, showing that only human EVeLNPs increased overall efficacy and lung tropism (FIG. 14). Example 4 - EVeLNPs increased protein expression in most lung cell types Transduction of different cell types by the EVeLNP formulations was examined. To do this, mRNA encoding Cre recombinase was delivered to Ai9 mice, which have a tdTomato reporter gene downstream of a Lox-Stop cassette. In naive mice, the Lox-Stop cassette prevents tdTomato expression, but when Cre recombinase is delivered into cells and translated, the stop cassette is removed, allowing for permanent expression of tdTomato. Such permanent expression allows for determination of which specific cell types were successfully transfected (Chaudhary et al., supra)51

[0080] 3060iio LNPs encapsulating mCre were delivered to Ai9 mice at a dose of 1 mg / kg mRNA and an EV protein concentration at 3.75 mg / kg. After three days, the mice were euthanized and tdTomato expression was measured with the IVIS®. Consistent with the results described above, a significant (10-fold) increase in tdTomato expression was observed in the lungs with the EVeLNPs compared to the LNPs. Expression in the other organs evaluated was not different when EVeLNPs and LNPs were compared (FIGS. 4A- 4B)

[0081] To determine which cell types were successfully transduced with each treatment, flow cytometry was used on dissociated liver and lung tissue. For most liver cell types, there was no change in total tdTomato expression. Interestingly, however, endothelial cells displayed reduced expression with the EVeLNPs compared to LNPs, with a 30% difference (FIG. 4C).

[0082] For the lung cell types examined, there was a significant increase in tdTomato expression in every cell type for EVeLNPs. The most significant increase in expression was observed in lung endothelial and epithelial cells (FIG. 4D). The highest increase occurred in vascular endothelial cells (VECs; -15% with LNP vs. -75% with EVeLNPs) and bronchial epithelial cells (BECs; -10% with LNP vs. -60% with EVeLNPs) (FIG. 4D). Lymphatic endothelial cells (LECs) and alveolar epithelial cells type 2 (AEC2) also showed substantially increased expression by about 40% for EVeLNPs vs. LNPs (FIG. 4D) In addition, lung immune cells displayed increased tdTomato expression, albeit to a lesser extent (FIG. 4E). Histology analysis was conducted to assess the safety of the particles in vivo. H&E staining did not show any inflammation of treated tissue compared to untreated controls (FIG. 4F).

[0083] To understand how the EVeLNPs increased lung delivery, studies were conducted to elucidate what was physically occurring between the EVs and LNPs when they were combined. Possibilities included: (1) that the LNPs and EVs were completely separate in the EVeLNPs, (2) that the EVs and LNPs were partially fused in the EVeLNPs, (3) that the EVs and LNPs were completely merged in the EVeLNPs, (4) that the EVs encapsulated LNPs in the EVeLNPs, and (5) that the LNPs encapsulated EVs in the EVeLNPs. Magnetic antibody sorting was used to characterize the EVeLNPs. Magnetic beads were coated with anti-CD9 (an EV marker), anti-PEG (an LNP marker), or isotype control antibodies. The LNPs were formulated by encapsulating fluorescent Cy5-labeled mRNA. The EVs, LNPs, and EVeLNPs were then mixed with the magnetic beads. Using flow cytometry, the Cy5 signal was quantified. These studies demonstrated that, as expected, the LNP-only sample attached to the anti-PEG antibody (positive control). Further, there was no Cy5 signal for the anti-CD9 antibody, comparable to the isotype control. The EV-only sample (not having any Cy5) matched the control for the anti-PEG and anti-CD9 (negative control). Interestingly, EVeLNPs showed signal in both anti-CD9 and anti-PEG (FIG. 15), meaning that the EVeLNPs contained both CD9 and PEG on the surface that encapsulated the labeled mRNA.

[0084] OTHER EMBODIMENTS

[0085] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. A composition comprising: a milk extracellular vesicle (EV), and a lipid nanoparticle (LNP) comprising a nucleic acid cargo.

2. The composition of claim 1, wherein the milk EV is from human breast milk.

3. The composition of claim 1 or claim 2, wherein composition comprises the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein.

4. The composition of any one of claims 1 to 3, wherein the LNP comprises an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound.

5. The composition of claim 4, wherein: the lipidoid is an ionizable lipid comprising 3060iio, the cholesterol or a derivative thereof is cholesterol, the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the PEG-based compound is PEG-2000.

6. The composition of any one of claims 1 to 5, wherein the composition comprises the LNP in an amount of about 0.1 ng / mL mRNA to about 100 pg / mL mRNA.

7. The composition of any one of claims 1 to 6, wherein the composition further comprises a pharmaceutically acceptable carrier.

8. The composition of claim 7, wherein the pharmaceutically acceptable carrier comprises one or more of water, saline, sucrose, dextrose, and trehalose.

9. The composition of any one of claims 1 to 8, wherein the nucleic acid comprises one or more of an mRNA, siRNA, saRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer.

10. The composition of any one of claims 1 to 8, wherein the nucleic acid comprises DNA.

11. A method for delivering a nucleic acid to a cell, wherein said method comprises contacting the cell with a composition comprising: a milk extracellular vesicle (EV), and a lipid nanoparticle (LNP) comprising a nucleic acid cargo.

12. The method of claim 11, wherein the milk EV is from human breast milk.

13. The method of claim 11 or claim 12, wherein composition comprises the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein.

14. The method of any one of claims 11 to 13, wherein the LNP comprises an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound.

15. The method of claim 14, wherein: the lipidoid is an ionizable lipid comprising 3060iio, the cholesterol or a derivative thereof is cholesterol, the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the PEG-based compound is PEG-2000.

16. The method of any one of claims 11 to 15, wherein the composition comprises the LNP in an amount of about 0.1 ng / mL mRNAto about 100 pg / mL mRNA.

17. The method of any one of claims 11 to 16, wherein the composition further comprises a pharmaceutically acceptable carrier.

18. The method of claim 17, wherein the pharmaceutically acceptable carrier comprises water, saline, sucrose, dextrose, or trehalose.

19. The method of any one of claims 11 to 18, wherein the nucleic acid comprises one or more of an mRNA, siRNA, saRNA, shRNA, miRNA, antisense RNA, guide RNA, long non-coding RNA, transfer RNA, ribosomal RNA, dsRNA, and an RNA aptamer.

20. The method of any one of claims 11 to 18, wherein the nucleic acid comprisesDNA.

21. The method of any one of claims 11 to 20, wherein the cell is in vitro, and wherein the method comprises contacting the cell with the composition in an amount of about 10 ng to about 1 pg mRNA and / or about 0.5 pg / mL to about 100 pg / mL EV protein.

22. The method of any one of claims 11 to 20, wherein the cell is within a mammal.

23. The method of claim 22, wherein the mammal is a human.

24. The method of claim 22 or claim 23, wherein the mammal has been identified as having an infectious disease, autoimmune disease, cardiovascular disease, respiratory disease, neurological disease, cancer, metabolic disease, musculoskeletal disease, hematologic disease, or any combination thereof.

25. The method of claim 22 or claim 23, wherein the mammal has been identified as having a condition or disease characterized by aberrant protein expression.

26. The method of any one of claims 22 to 25, wherein the contacting comprises contacting the cell with the composition in an amount of about 0.05 to about 5 mg / kg mRNA and / or about 0.1 to about 10 mg / kg EV protein.

27. A method for delivering a messenger RNAto a cell within a mammal, wherein said method comprises administering, to the mammal, a composition comprising: a milk extracellular vesicle (EV), and a lipid nanoparticle (LNP) comprising a messenger RNA (mRNA) cargo.

28. The method of claim 27, wherein the milk EV is from human breast milk.

29. The method of claim 27 or claim 28, wherein the composition comprises the milk EV in an amount of about 0.1 mg / mL protein to about 15 mg / mL protein.

30. The method of any one of claims 27 to 29, wherein the LNP comprises an ionizable lipid or lipidoid, cholesterol or a derivative thereof, a helper lipid, and a polyethylene glycol (PEG)-based compound.

31. The method of claim 30, wherein: the lipidoid is an ionizable lipid comprising 3060iio, the cholesterol or a derivative thereof is cholesterol,the helper lipid is l,2-dioleoyl-sn-glycero-3-phosphoethanolamine, and the PEG-based compound is PEG-2000.

32. The method of any one of claims 27 to 31, wherein the composition comprises the LNP in an amount of about 0.1 ng / mL mRNAto about 100 pg / mL mRNA.

33. The method of any one of claims 27 to 32, wherein the composition further comprises a pharmaceutically acceptable carrier.

34. The method of claim 33, wherein the pharmaceutically acceptable carrier comprises water, saline, sucrose, dextrose, or trehalose.

35. The method of any one of claims 27 to 34, wherein the mammal is a human.

36. The method of any one of claims 27 to 35, wherein the administering comprises administering the composition to the mammal in an amount of about 0.05 to about 5 mg / kg mRNA and / or about 0.1 to about 10 mg / kg EV protein.

37. The method of any one of claims 27 to 36, wherein the cell is a lung cell.

38. The method of any one of claims 27 to 37, wherein the composition is administered intravenously, intramuscularly, intraperitoneally, intrathecally, nasally, by inhalation, orally, or subcutaneously.

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