Lung-specific mRNA delivery enabled by sulfonium lipid nanoparticles

Abstract

Among various mRNA carrier systems, lipid nanoparticles (LNPs) stand out as the most clinically advanced. While current clinical trials of mRNA / LNP therapeutics mainly address liver diseases, the potential of mRNA therapy extends far beyond. To fully unlock the promises of mRNA therapy, there is an urgent need to develop safe and effective LNP systems that can target extrahepatic organs. Embodiments disclosed herein, are directed to novel sulfonium lipid nanoparticles (sLNPs) for systemic mRNA delivery to the lungs. sLNP effectively and specifically deliver mRNA to the lungs following intravenous administration in mice. No evidence of lung and systemic inflammation or toxicity in major organs was induced by sLNP. Our findings demonstrated that the newly developed lung-specific sLNP platform is both safe and efficacious and provides for the development of new mRNA-based therapies for the treatment of lung-associated diseases and conditions.

Description

LUNG-SPECIFIC mRNA DELIVERY ENABLED BY SULFONIUM LIPID NANOPARTICLESFIELD OF THE INVENTION

[0001] The present disclosure is generally in the fields of organic chemistry and medicine. More particularly, it concerns therapeutic and preventative agents for treating or preventing diseases, and disorders, particularly of the lungs. In embodiments, the disclosure relates to identifying therapeutic and preventative agents for treating diseases, disorders and conditions of the lungs.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of the filing date of U.S. Provisional Application No. 63 / 562,409, filed March 7, 2024. The content of this earlier filed application is hereby incorporated by reference herein in its entirety.BACKGROUND

[0003] mRNA therapeutics hold great promise in protein replacement therapy, gene editing, oncology, etc. Lipid nanoparticles (LNPs) are the most clinically advanced delivery system for RNA drugs. In addition to vaccines, LNP-enabled mRNA therapeutics are undergoing thorough investigation across various fields. Despite the great efforts, however, the ongoing clinical trials of mRNA / LNP therapeutics mainly focus on liver-associated diseases. An efficacious LNP system that can target extrahepatic organs is the key to unlocking the potential of mRNA therapeutics.

[0004] Lung-targeted mRNA delivery is useful in treating various lung-associated diseases, such as cystic fibrosis and lung cancer. LNPs have been employed for both local, e.g. inhalation, and intratracheal, as well as for systemic, e.g. intravenous delivery of mRNA to the lungs. Local administration is straightforward and efficient, but when challenges arise, e.g. toxicity, irritative reactions, and patient inability, systemic delivery can be preferred.

[0005] For systemic mRNA delivery to the lung, several LNP systems have been developed. Kaczmarek et al. synthesized a library of polymer-lipid nanoparticles for the delivery of mRNA and DNA. Their nanoparticles primarily delivered mRNA to thelungs following intravenous injection and a strong protein production was observed within 48 hours post-injection. Cheng and colleagues reported a systematic approach for fabricating lung-targeting LNP systems. Their versatile strategy incorporated a proper amount of cationic excipient lipid (e.g. DOTAP) into the four-component ionizable lipid-containing formulations, and results show LNP redirection from the liver to the lungs after systemic injection. Later, LoPresti et al. demonstrated that simply replacing the helper phospholipid in the four-component LNP formulation with permanently charged cationic lipids also facilitates lung targeting. More recently, Qiu et al. reported on a group of amide linker-containing LNPs for lung-selective delivery of mRNA and showed therapeutic effects in a mouse model of lymphangioleiomyomatosis.

[0006] There is an urgent need to develop safe and effective LNP systems that can target extrahepatic organs. Embodiments disclosed herein, are directed to novel sulfonium lipid nanoparticles (sLNPs) for systemic mRNA delivery to the lungs.SUMMARY OF THE INVENTION

[0007] Embodiments disclosed herein are directed to a sulfonium lipid compound having a formula (R1 )(R2)(R3)S+X-, wherein R1 , R2, and R3 are organic groups, S+ is the positively charged sulfur atom, and X- is a negatively charged counterion, and at least one of R1 , R2, and R3 groups contain at least one hydrophilic moiety and at least one of R1 , R2, and R3 groups contain at least one hydrophobic moiety. According to an embodiment the hydrophilic moiety is at least one selected from the group consisting of hydroxyl, carboxyl, ether, amino, amide, amino acid, phosphonate, sulfate, sulfonate, carbohydrate, and carbonyl; and the hydrophobic moiety is at least one selected from the group consisting of linear or non-linear alkyl, phenyl, alkenyl, sterol, halogen, halogen ether, and oligo- or poly-ester, ether, amide, carbonate, urea, and urethane.

[0008] According to an embodiment the negatively charged counterions is at least one selected from the group consisting of Chloride (CI-), Bromide (Br-), Iodide (I-), Phosphate (PO4Λ3-), Carbonate (CO3Λ2-), Sulfate (SO4Λ2-) tetrafluoroborate (BF4-), trifluoromethanesulfonate (CF3SO3-), methanesulfonate (CH3SO3-), and tosylate (CH3C6H6SO3-). According to an embodiment the negatively charged counterions is DNA and / or RNA.

[0009] According to an embodiment the sulfonium lipid compound is DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium), or DOSEH ((2-hydroxyethyl)dioctadecyl sulfonium)), or HOSEH (hexadevyl(2-hydroxyethyl)octadecyl sulfonium), or DHSEA ((2-amino-2-oxoethyl)dihexadecyl sulfonium), or DOSPH ((3- hydroxypropyl)dioctadecyl sulfonium).

[0010] According to other embodiments herein, a composition for lung-specific mRNA delivery is disclosed. The composition comprises a) a sulfonium lipid nanoparticle (sLNP); and b) an mRNA payload encapsulated within the sLNP, and the sulfonium lipid nanoparticle is at least one selected from the group consisting of DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium), DOSEH ((2- hydroxyethyl)dioctadecyl sulfonium)), HOSEH (hexadevyl(2-hydroxyethyl)octadecyl sulfonium), DHSEA ((2-amino-2-oxoethyl)dihexadecyl sulfonium), and DOSPH ((3- hydroxypropyl)dioctadecyl sulfonium). According to embodiments the composition’s sulfonium lipid nanoparticle has a size range of about 50 nm to about 500 nm and a positive zeta potential. According to embodiments the sLNP comprises a mixture of sulfonium lipids, a helper lipid, sterol, and PEG-lipids and the helper lipid is at least one selected from the group consisting of DSPC (distearoylphosphatidylcholine), DOPC (1 ,2-Dioleoyl-sn-glycero-3-phosphocholine), DMPC (1 ,2-Dimyristoyl-sn- glycero-3-phosphocholine), DPPC (1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1 ,2-dioleoyl-sn- glycero-3-phosphocholine), DOPS (1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine), phosphatidylserine, and DOTAP (1 ,2-dioleoyl-3-trimethylammonium-propane).According to embodiments, the sterol of the composition is at least one selected from the group consisting of cholesterol, bile acid, β-Sitosterol, Stigmasterol, Fucosterol, Ergosterol, Fungisterol, 7-Dehydrocholesterol, Saringosterol. According to embodiments, the mRNA encapsulation efficacy is greater than 80% within the composition. According to embodiments the sLNP demonstrates higher specificity for lung endothelial cells compared to other lung-targeting lipid nanoparticles, and the sLNP maintains mRNA stability in bloodstream circulation prior to lung-specific delivery.

[0011] According to embodiments disclosed herein are methods for delivering mRNA to lung tissue. The methods comprising administering the claimed composition intravenously to a subject in need thereof, wherein the method results in delivery of mRNA to the lungs without evidence of lung and systemic inflammation ortoxicity in major organs and the mRNA is delivered to endothelial, epithelial, and immune cells, and wherein the pulmonary endothelial cells are primary targets. According to other embodiments are methods for treating a lung-associated disease or condition, which comprises administering a therapeutically effective amount of the claimed composition to a subject in need thereof. The mRNA of the method encoding a therapeutic protein for treating a lung-associated disease.

[0012] Other embodiments disclosed herein are directed to a sulfonium lipid nanoparticle (sLNP) composition for delivering mRNA to the lungs. The composition comprises at least one sulfonium lipid, with or without one helper lipid, with or without sterol derivative; and with or without one PEG-lipid, and the delivered mRNA encodes a therapeutic protein for treating a lung-associated disease. Other embodiments are directed pharmaceutical compositions for lung-specific mRNA delivery, comprising: a) an sLNP composition; b) a therapeutic mRNA; and c) a pharmaceutically acceptable carrier. The sLNP of the pharmaceutical composition demonstrates lung-specific mRNA delivery following intravenous administration without evidence of lung inflammation or systemic toxicity.

[0013] As such, embodiments disclosed herein are directed to novel sulfonium lipid nanoparticles (sLNPs), use of sulfonium lipid nanoparticles (sLNPs) for delivering mRNA to the lungs of a subject, and methods of making and using the disclosed sLNPs.The disclosed sulfonium lipid nanoparticles differ from other lipid nanoparticles in several key ways. For example, the disclosed lipid composition, i.e., sLNPs are constructed using novel sulfonium lipids, which feature positively charged sulfonium groups for mRNA complexation and intracellular delivery, contrary to the conventional amine-based cationic and ionizable lipids. The sLNPs demonstrate effective and specific delivery of mRNA to the lungs following intravenous administration in mice. The sulfonium lipids (for example, DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium), DOSEH ((2-hydroxyethyl)dioctadecyl sulfonium)), HOSEH (hexadevyl(2- hydroxyethyl)octadecyl sulfonium), DHSEA ((2-amino-2-oxoethyl)dihexadecyl sulfonium), and DOSPH ((3-hydroxypropyl)dioctadecyl sulfonium) used in sLNPs have a distinct chemical structure that enables self-assembly with mRNA and targeted delivery to the lungs. Further, there is no evidence of lung and systemic inflammation or toxicity in major organs by sLNP, indicating a favorable safety profile. The sLNPs showed higher transfection efficacy in bulk lung and pulmonaryendothelial cells compared to benchmark lung-targeting LNPs. As such, these unique characteristics make sLNPs a promising new class of carrier system for systemic delivery of mRNAto the lungs, with potential applications in treating lung- associated diseases and conditionsBRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 presents sulfonium lipid nanoparticle (sLNP)-enabled systemic delivery of mRNAto the lungs. (A) is a schematic illustration of the chemical structures of sulfonium lipids (DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium) and DOSEH (2-hydroxyethyl)dioctadecyl sulfonium)), the self-assembly process of mRNA-loaded sLNP (mRNA / sLNP), and the targeted delivery of mRNAto the lungs in mice. (B) is a representative in vivo whole-body bioluminescence images of mice treated with PBS, free fLuc mRNA, and mRNA / sLNPs. (C) is a representative ex vivo bioluminescence images of mouse organs. (D) presents a quantification of total bioluminescence flux from the lungs of mice treated with PBS, free mRNA, and mRNA-loaded LNPs. Data are presented as mean ± s.e.m., n = 3. (E) is the percentage of background-subtracted bioluminescence signal originating from each organ in mice treated with mRNA-loaded LNPs.

[0015] FIG. 2 presents distribution of protein expression and kinetics study of mRNA / sLNP. (A) is a schematic illustration of mouse lung lobes. (B) is representative ex vivo bioluminescence images of dissected lung lobes. (C) presents a quantification of average radiance for all five lung lobes. Data are presented as mean ± s.d., n = 3, one-way ANOVA analysis. (D) is a whole-body bioluminescence images and (E) is a graph quantification of total flux at different time points for high (0.4 mg / kg) and low doses (0.1 mg / kg). Data are presented as mean ± s.d., n = 3. (F) is a graph of the area under the curve (AUC) analysis shows cumulative bioluminescence.

[0016] FIG. 3 presents sLNP-mediated mRNA delivery for genome engineering in the mouse lungs. (A) is a schematic illustration of Cre-Lox recombination and tdTomato activation in Ai14 mouse following successful Cre mRNA delivery. (B) is a representative ex vivo fluorescence images of lungs. (C) is a representative confocal images and (D) is a quantification of tdTomato-positive cells per field of view (FOV) of lung slices obtained from mice received PBS, Cre mRNA / sLNP, or Cre mRNA / MC3- DOTAP. Scale bar = 100 urn. (E) presents flow cytometry quantification of tdTomato-positive cells within lung endothelial, epithelial, and immune cell populations. Data are presented as mean ± s.d., n = 3, two-tailed unpaired t-test.

[0017] FIG. 4 presents proteomics analysis of sLNP protein corona. (A) are the top 10 most abundant proteins on corona. The enriched proteins on the corona were categorized based on their (B) isoelectric point, (C) molecular weight, and (D) biological function.

[0018] FIG. 5 presents data indicating that sLNP does not induce inflammation or toxicity in mice. (A) presents representative images of cells collected in BALE (B) is a quantification of macrophage counts, (C) presents variations in cytokine levels, and (D) the total protein concentration in bronchoalveolar lavage fluid (BALF). (E) presents cell counts of red blood cell (RBC) and (F) white blood cell (WBC). (G) presents quantification of AST and (H) BUN in serum. Data are presented as mean ± s.d., n = 3, two-tailed unpaired t-test. (I) presents H&E staining images of major organ slices. Scale bar = 100 pm.

[0019] FIG. 6 presents (A)1H NMR and (B) MS spectra of DHSEH.

[0020] FIG. 7 presents (A)1H NMR and (B) MS spectra of DOSEH.

[0021] FIG. 8 presents ex vivo bioluminescence images of organs obtained fromBalb / c mice treated with fLuc mRNA / MC3-DOTAP.

[0022] FIG. 9 presents a characterization of mRNA / sLNP formulations. (A) presents an average hydrodynamic diameter (<Dh>), (B) polydispersity (PDI), (C) zeta-potential, (D) mRNA encapsulation efficacy, and (E) representative cryoEM images of mRNA / DHSEH and mRNA / DOSEH. Scale bar = 100 nm. Data are presented as mean ± s.d., n = 3.

[0023] FIG. 10 presents an ex vivo bioluminescence images of mouse organs 72 h post-administration of mRNA / sLNP.

[0024] FIG. 11 presents FACS gating strategy for analysis of tdTomato+ lung cells. PBS-treated mice were sacrificed, and single-cell suspension of lung cells was prepared for flow cytometry analysis.

[0025] FIG. 12 presents FACS gating strategy for analysis of tdTomato+ lung cells. mRNA / sLNP-treated mice were sacrificed, and single-cell suspension of lung cells was prepared for flow cytometry analysis.

[0026] FIG. 13 presents (A) Lymphocyte, monocyte, and neutrophile cell counts and (B) white blood cell differentials.

[0027] FIG. 14 presents (A) body weight measurements, (B) serum ALT activity, and (C) relative concentration of creatinine in PBS- and mRNA / sLNP-treated mice.

[0028] FIG. 15 presents the chemical structures of four groups of sulfonium lipids and their lung-targeted mRNA delivery in vivo in mice. Sulfonium lipids with (A) different tail length, (B) asymmetric tails, (C) head groups, and (D) linker types. In a firefly luciferase mRNA reporter system, total bioluminescence flux from the lungs are quantified and representative MS images are shown. Data are presented as mean±s.e.m., N = 3.DEFINITIONS

[0029] Lipids as disclosed herein are a diverse group of organic compounds that are relatively insoluble in water but soluble in nonpolar and polar organic solvents such as benzene, chloroform, ether, methanol, ethanol, dimethyl sulfoxide, dichloromethane, ethyl acetate, acetonitrile, and acetone. Lipids are hydrophobic or amphiphilic in nature, and they are either completely hydrophobic or have both hydrophobic and hydrophilic parts (amphiphilic). Natural lipids play roles in energy storage, cell signaling, and forming structural components of cell membranes. The lipid category encompasses fats, oils, waxes, sterols, fat-soluble vitamins, phospholipids, and other related compounds. Lipids contain hydrogen, carbon, and oxygen atoms, which form the framework for the structure and function of living cells. Lipids are a broad classification of compounds that includes various types of molecules with similar physical properties, despite their diverse structures and functions in biological systems.

[0030] A sulfonium lipid compound as disclosed herein is a type of fully synthetic, cationic lipid that contains a positively charged sulfonium ion (a sulfur atom bonded to three organic groups) as its head group. These compounds comprise a sulfonium head group, a positively charged sulfur atom bonded to three organic substituents, an alkyl linker connecting the hydrophilic group to the lipid, a aliphatic chain(s) or aromatic group(s), and a hydrophobic tail(s) that contribute to the lipid structure.

[0031] A helper lipid as disclosed herein is DOPC (1 ,2-Dioleoyl-sn-glycero-3- phosphocholine), a crucial component of the lipid nanoparticle (LNP) formulations used for drug delivery, particularly for nucleic acid therapeutics like siRNA and mRNA. Helper lipids contribute to the formation, stability, and functionality of LNPs in several ways. Some other examples of helper lipids are DSPC(distearoylphosphatidylcholine) and DOPE (1 ,2-dioleoyl-sn-glycero-3- phosphoethanolamine). Cholesterolincreases membrane rigidity and , stabilize lipid nanoparticle structures, and facilitate cell uptake. The proportion and type of helper lipids, cholesterol, and PEG-lipids in LNP formulations can be adjusted to optimize the delivery system's performance for specific applications in drug delivery and gene therapy.

[0032] A compound as disclosed herein is a pure substance composed of two or more different elements chemically bonded together in a fixed ratio.

[0033] A composition as disclosed herein refers to the arrangement, ratio, and type of atoms or substances in a material.

[0034] As disclosed herein negatively charged counterions play a crucial role in balancing the charges of positively charged sulfonium lipid and maintains the overall electrical neutrality of sulfonium lipid assemblies. Examples of negatively charged counterions include: Chloride (CI-): This is a common monovalent anion that often serves as a counterion for cationic lipids; Phosphate (PO4Λ3-), wherein the phosphate group is a polyvalent anion that can act as a counterion, particularly in biological systems where it's abundant; Carbonate (CO3A2-) is a divalent anion can serve as a counterion in certain lipid systems; Sulfate (SO4Λ2-) is another divalent anion that can function as a counterion for positively charged lipids. Other anionic counterion include tetrafluoroborate (BF4-), trifluoromethanesulfonate (CF3SO3-), methanesulfonate (CH3SO3-), and tosylate (CH3C6H6SO3-) In the formation of lipoplexes (cationic lipid-DNA / RNA complexes), the negatively charged phosphate backbone of DNAand RNA acts as a counterion to cationic lipids. These negatively charged counterions interact with positively charged lipid head groups, influencing the lateral distribution of lipids within membranes and affecting properties such as membrane stability, curvature and fluidity. The release of these counterions during lipid-macromolecule interactions can also drive various binding processes, including protein-membrane and DNA-membrane complexation.

[0035] A consistent protein expression pattern, as disclosed herein is directed to the mRNA-encoded protein production in the lungs during the whole 72 hours experimental period. No re-distribution in other organs was found. It means our sLNP delivered mRNA stayed in the lungs the whole time.

[0036] Total protein production as disclosed herein is directed to the bioluminescence signal to semi-quantify how much protein was produced over the experimental period.

[0037] Zeta potential as disclosed herein relates to colloidal chemistry and measures the electrical potential difference between the bulk of a liquid and the stationary layer of fluid attached to a dispersed particle. It is typically expressed in millivolts (mV) and represents the electrokinetic potential at the slipping plane of a particle in suspension and indicates the degree of electrostatic repulsion between similarly charged particles in a dispersion. Higher absolute values of zeta potential (>30 mV) generally indicate better stability, while lower values suggest a tendency for particles to aggregate.

[0038] As disclosed herein, lung-associated disease or condition include: Idiopathic Pulmonary Fibrosis (IPF); Inflammatory Pulmonary Fibrosis; Asthma; Chronic Obstructive Pulmonary Disease (COPD); Interstitial Lung Disease (ILD); Respiratory Infections, Alpha-1 -antitrypsin deficiency; and Pulmonary Hypertension.DETAILED DESCRIPTION

[0039] Continued efforts are essential for the development of novel, safe, and effective (LNP) systems tailored for lung-specific mRNA delivery. Embodiments disclosed herein are directed to the synthesis of a novel class of LNPs, namely sulfonium LNPs (sLNPs), constructed from sulfonium lipids, and their utilization for delivering mRNA systemically to the lungs. In contrast to conventional amine-based lipids, our newly developed lipids feature positively charged sulfonium groups for mRNA complexation and intracellular delivery. Embodiments disclosed herein are directed to sulfonium lipids and sLNP developed specifically for mRNA delivery and organ targeting. Our findings demonstrate that upon intravenous administration, the sLNPs efficiently and safely deliver various types of mRNA to the lungs, leading to robust protein production (FIG. 1A).

[0040] The synthesis of novel sulfonium lipids and sLNPs, such as the sulfonium lipids DHSEH, DOSEH, HOSEH (hexadevyl(2-hydroxyethyl)octadecyl sulfonium), DHSEA ((2-amino-2-oxoethyl)dihexadecyl sulfonium), and DOSPH ((3- hydroxypropyl)dioctadecyl sulfonium), are identified herein (see FIG. 1 and FIG. 15, respectively) and were synthesized according to the procedure more fully describedherein below. The chemical structures of DHSEH and DOSEH were characterized by1H NMR and MS (FIG. 6 and FIG. 7).

[0041] The sLNPs containing sulfonium lipids (DHSEHorDOSEH), cholesterol, 1,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol (DMG-PEG2k) were fabricated through self-assembly (FIG. 1A). The sulfonium lipids provide a matrix to incorporate other ingredients, and they can bind with mRNA molecules primarily through electrostatic interactions. The sulfonium compound can be DHSEH, DOSEH, HOSEH, DHSEA, DOSPH, and the like. Cholesterol is used to stabilize the supramolecular structure, tune the rigidity and fluidity of the lipid membrane, and promote cellular internalization. The helper phospholipids (e.g., DOPC) participate in the formation of the lipid bilayer and facilitate endosomal escape. PEG-lipid is used to improve stability by providing steric hindrance and non-fouling characteristics. In addition to the example described above, the sLNP formulation can consist of a wide variety of sulfonium and excipient lipids. For example, the sterol can be naturally occurring, semi- or fully-synthetic, such as cholesterol, sitosterol, dexamethasone, and the like. The phospholipid can be DOPC (1 ,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE (1,2-dioleoyl-sn-glycero- 3-phosphoethanolamine), DSPC (1 ,2-distearoyl-sn-glycero-3-phosphocholine), and others. The PEG-lipid can be PEG-DMG (1 ,2-dimyristoyl-rac-glycero-3- methoxypolyethylene glycol), PEG-DSPE (1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-carboxy(polyethylene glycol)), and others, and the molecular weight of PEG can be 500 Da to 10000 Da. The molar percentage of each component (i.e. sulfonium lipids, sterol, phospholipid, PEG-lipid) can vary from 0.1% to 99.9%.

[0042] With respect to the sLNP-enabled lung-specific delivery of mRNA we utilized in vivo mRNA delivery studies, wherein fLuc (firefly luciferase) mRNA was loaded into sLNPs (denoted as mRNA / DHSEH and mRNA / DOSEH). Balb / c mice received either PBS, free mRNA, or mRNA / sLNP (0.1 mg mRNA / kg body weight). Both mRNA / DHSEH and mRNA / DOSEH induced strong bioluminescence signals, suggesting successful mRNA delivery and protein production (FIG. 1B). The observed signals primarily originate from the lungs. Previous studies have shown that positively charged LNPs tend to accumulate in the lungs following intravenous injection. Dilliard et al. showed that a group of lung-specific LNPs shared similar apparent pKa values of >9.25, suggesting that these LNPs possess net positivecharges at physiological pH. In another study, Huang et al. showed that the quaternization of amine lipids redirected LNP from the spleen to the lungs. No signals were recorded for mice injected with PBS or free mRNA (FIG. 1 B), indicating systemic administration of free mRNA cannot induce efficacious protein expression. Consistent with the in vivo imaging results, ex vivo bioluminescence signals were predominantly observed in the lungs of mice receiving mRNA / sLNPs (FIG. 1 C). The total bioluminescence emission flux from lungs was then quantified (FIG. 1 D). Lungs from mice receiving free mRNA (ca. 3.49 x 104p / s) showed a similar signal intensity compared to the PBS group (ca. 4.51 x 104p / s), further confirming the inefficiency of systemically injected free mRNA. mRNA / DHSEH led to ~200-fold intensity increase (ca. 7.25 x 106p / s) compared to the free mRNA group, and mRNA / DOSEH was more efficient, showing a 2.79-fold higher flux intensity compared with mRNA / DHSEH (ca. 2.02 x 107p / s). Notably, the mRNA / sLNP formulations exhibited stronger signals compared to the benchmark lung-targeting MC3-DOTAP LNPs (ca. 1.18 x 105p / s; FIG. 1 D and FIG. 8). In terms of organ specificity (FIG. 1 E), it was found that ca. 95.3% and 96.5% bioluminescence signals originated from the lungs of mice administered with mRNA / DHSEH and mRNA / DOSEH sLNP, respectively, outperforming the MC3-DOTAP formulation (ca. 88.8%).

[0043] The physicochemical properties of mRNA / sLNP were characterized. mRNA / DHSEH and mRNA / DOSEH showed average hydrodynamic diameters (<Dh>) of 243.6 and 188.3 nm, respectively (FIG. 9A). Their sizes are in the optimal range for systemic circulation, as previous studies indicated that small nanoparticles (e.g. <10 nm) could undergo renal clearance, and big LNPs (e.g. >400 nm) are prone to hepatic clearance. It should be noted that, apart from the size, other physicochemical traits (e.g. surface charge and chemistry) all affect LNPs' behaviors. Both mRNA / DHSEH (0.184) and mRNA / DOSEH (0.229) had a PDI lower than 0.3, implying their uniformity (FIG. 9B). Both mRNA / DHSEH (42.52 mV) and mRNA / DOSEH (46.38 mV) were positively charged, attributing to the cationic sulfonium moieties (FIG. 90). The mRNA encapsulation efficacy was determined to be >99% for both DHSEH and DOSEH sLNPs (FIG. 9D). We speculated that the cationic nature of sulfonium lipids and high positive charge density contributed to efficient mRNA binding. Finally, the morphology of sLNPs was examined using cryoEM. Both formulations showed a similar spherical liposomal structure, in which both unilamellar and multilamellar vesicles were observed (FIG. 9E).

[0044] Vesicles are one of the most common self-assembly structures of natural and synthetic lipids, and they have been extensively studied for the delivery of small molecule drugs, proteins, and nucleic acids. Consistent with the well-studied siRNA / LNP systems, it was envisioned that the negatively charged mRNA molecules can bind to the inner and exterior lipid bilayer surfaces mainly through electrostatic interactions, and they can also be sandwiched between two layers in the multilamellar vesicles. The physical complexation improves the stability of mRNA molecules during systemic circulation and facilitates extravasation and cell internalization.

[0045] A common issue associated with the local drug administration to lungs is the suboptimal distribution of drugs. In this respect, we utilized lung distribution and kinetics studies. In one study, locally administered viral vector-induced protein expressions were primarily found in the nasal cavity, trachea, and proximal aspects of the lower respiratory tract. Little to no protein production was found at the mid to distal aspects of lung lobes. Systemic delivery can potentially overcome this limitation as drugs are carried and distributed by systemic blood circulation. We dissected the lung lobes of Balb / c mice receiving fLuc mRNA / DOSEH (FIG. 2A). All five lobes (i.e. right cranial (RCr), right middle (RMd), accessory (RAc), right caudal (RCd), and left) showed strong bioluminescence emission, and both the proximal and distal sites of primary bronchi had similar emission intensity (FIG. 2B). Furthermore, bioluminescence radiance (p / s / cm2 / sec) was quantified, and there was no significant difference between five lobes (FIG. 2C). For kinetics study, fLuc mRNA / DOSEH was injected into Balb / c mice, and in vivo whole-body bioluminescence images were obtained at certain time intervals within 72 h (FIG. 2D). At a dose of 0.1 mg mRNA / kg body weight, the signal gradually increased from 3 h to 6 h (FIG. 2E) and followed by a graduate decrease. No signals above the background were recorded at 72 hours, indicating the exhaustion of delivered mRNA. This is comparable with the mRNA delivery kinetics observed for polymer-lipid nanoparticle D90-C12-103 reported in previous study. We then speculated that if a higher dose would induce longer and greater protein expression. At a dose of 0.4 mg mRNA / kg body weight, the signal peaked at 6 hours, and then gradually decreased. At the endpoint of this experiment, 72 hours, the emission signal was still above the background level. Mice were then sacrificed, and ex vivo organ imaging further confirmed bioluminescence emission from the lungs, and no signals were recorded for other organs (i.e. heart, liver, spleen, kidney; FIG. 10). A consistent protein expression pattern further demonstrated thelung selectivity of our mRNA / sLNP system, as no obvious re-distribution of delivered mRNA or encoded protein was found. The total protein production was calculated as the accumulated luminescence, and the 0.4 mg / kg group showed a ca. 90.4-fold higher protein production compared to the 0.1 mg / kg dose group (FIG. 2F).

[0046] We also examined the possibility of using sLNP for the delivery of Cre recombinase mRNA for genome engineering in the lungs. Ai14 mice were used, which contain a loxP-floxed STOP codon upstream of the tdTomato reporter gene in its genome (FIG. 3A). Cre mRNA / DOSEH was administered to Ai14 mice (0.42 mg / kg), and lungs were harvested five days post-injection. Compared to the PBS- treated group, red fluorescence signals were observed in the lungs treated with Cre mRNA / sLNP (FIG. 3B). Strong red fluorescence signals were recorded for both Cre mRNA / sLNP and the lung-tropic mRNA / MC3-DOTAP-injected mouse lung tissue slices (ca. 10 pm), and tdTomato-positive cells were found to be present evenly in the lungs (FIG. 3C). Further analysis revealed that sLNP induced approximately 2.69 times more tdTomato-positive cells / FOV, highlighting its high efficacy (FIG. 3D). To further illustrate the identity of mRNA-transfected and tdTomato-positive cells in the lungs, single-cell suspensions were then prepared and stained with antibodies (i.e. anti-CD31 , anti-CD326, and anti-CD45) for flow cytometry analysis. It was revealed that ca. 67.0% endothelial cells, 11.3% epithelial cells, and 3.5% immune cells were successfully transfected (FIG. 3E). Typical gating strategies for flow cytometry analysis are summarized (FIG. 11 and FIG. 12). The lung-tropic MC3-DOTAP induced approximately 41.0%, 17.3%, and 1.7% tdTomato-positive endothelial, epithelial, and immune cells, respectively. While sLNPs and MC3-DOTAP showed similar efficiency in transfecting epithelial cells, sLNPs were significantly more effective in transfecting endothelial and immune cells in the lungs (FIG. 3E). In the work by Qiu et al., 306-N16B transfected ca. 33.6% endothelial cells, 1.5% epithelial cells, and 1.9% immune cells. Another formulation with 113-N16B transfected 69.6% endothelial cells, 7.3% epithelial cells, and 18.9% immune cells. Cheng et al. reported the efficiency of ca. 66% endothelial cells, 39% epithelial cells, and 21 % immune cells in their SORT LNP study. Furthermore, an estimated 22% endothelial cells, 4% epithelial cells, and 7% immune cells were observed in a polymer-assisted five-element nanoparticle system developed by Cao et al. Taken together, our findings indicated that the sLNP demonstrated a transfection efficacy in lung endothelial cells comparable to that of the benchmark lung-targeting LNPs.Furthermore, our results suggested that the sLNP displayed a higher specificity for endothelial cells, which could be advantageous in scenarios where the lung endothelium is the primary target for mRNA therapeutics delivery.

[0047] It was expected that a protein corona would form on the surface of LNPs immediately after administration in the bloodstream. Previous studies suggested a close relationship between the property of protein corona and LNPs’ biodistribution profile. To identify the protein corona on sLNP, we incubated DOSEH sLNP with adult C57BL / 6 mouse plasma and performed mass spectrometry proteomics analysis. A total of 245 different types of protein species were found adsorbed to DOSEH sLNP. The top ten most abundant proteins constitute -52.3% of total proteins on the corona (FIG. 4A). Among these adsorbed serum proteins, fibrinogen alpha chain (FIBA), serine protease inhibitor A3K (SPA3K), and apolipoprotein A-1 (APOA1 ) are the top three. Since the sLNPs are positively charged, we envisioned plasma proteins with net negative charges under physiological pH could be more easily adsorbed onto sLNPs. It was found that -72.2% of proteins possess a isoelectric point (PI) lower than 7.4, indicating the negatively charged proteins are preferred for sLNP binding (FIG. 4B). Around 27.8% proteins have a PI > 7.4, suggesting that apart from the electrostatic interactions, some other types of supramolecular interactions are involved for non-specific protein binding, such as hydrogen-bonding and hydrophobic interactions. Next, the molecular weights of corona proteins were analyzed (FIG. 4C). It was found that proteins with a molecular weight less than 100 kDa constitute ca. 80.8% of the corona, and most of the enriched proteins (abundance of - 25.7%) are in the range of 40-60 kDa. Finally, the corona proteins were further categorized according to their biological functions (FIG. 4D). Complement proteins (ca. 30.9%) and coagulation proteins (ca. 29.1%) are the two most abundant in the corona, followed by lipoproteins (ca. 13.4%) and immunoglobulins (ca. 2.9%). Furthermore, the top twenty most abundant corona proteins, which constitute around 72.2% of the corona, were summarized, along with their molecular weight, PI, and average abundance (Table 1 ). A comparison between sLNP protein corona with previously reported LNPs’ corona revealed that certain protein species (e.g. fibrinogens, apolipoproteins, fibronectin, vitronectin, and complement proteins) were commonly shared between these lung-targeting LNPs. Previous studies suggested that vitronectin might play important roles in the internalization of LNPs by lung cells. Particularly, the in vitro vitronectin-coating augmented cell transfection in avp3integrin-expressing cell lines. It is noteworthy that alpha-1 -antitrypsin (AAT) is one of the top protein species enriched in the sLNP corona (Table 1 ). Considering the protective function of AAT in the lungs and the fact that AAT can be internalized by lung endothelial cells via endocytosis, the role of AAT in mediating sLNP lung targeting merits further investigation.

[0048] We noted that mRNA / sLNP does not induce local or systemic inflammation and toxicity. Safety and biocompatibility are critical parameters in evaluating the translational potential of an mRNA delivery system. A recent study revealed that the cationic LNP formulation incorporated with DOTAP induced thrombosis in the lung and other organs. We first examined the possible local inflammation reaction in the lungs induced by mRNA / sLNP. The bronchoalveolar lavage fluid (BALF) was collected from Balb / c mice receiving mRNA / DOSEH. Similar numbers of macrophages in BALF were found in the PBS- and mRNA / sLNP-treated mice (Fig. 5A and FIG. 5B), and no neutrophil was observed. The mRNA / sLNP did not result in significant variation in major cytokine expression (i.e. TNF-a, IL-1 β and IL-6; FIG. 5C). Furthermore, no significant difference in total protein concentration was observed (FIG. 5D). These results suggested that the mRNA / sLNP did not induce obvious lung inflammation. Next, the potential hematological toxicity was examined. The total counts of red blood cells (RBCs; FIG. 5E), white blood cells (WBCs; FIG. 5F), lymphocytes, monocytes, and neutrophils (FIG. 13A) in blood collected from mRNA / sLNP-treated mice showed no significant difference compared to the PBS- treated control group. Furthermore, both the mRNA / sLNP and PBS-treated groups possessed similar WBC differentials (FIG. 13B). Next, the mouse body weight was monitored continuously for five days, and both mRNA / sLNP and PBS-treated mice showed a negligible body weight variation in the time course of this study (FIG. 14A). No obvious clinical signs of toxicity were recorded (e.g. impeded movement, aberrant behavior, etc.). The potential hepatotoxicity and nephrotoxicity were evaluated by measuring the blood concentrations of AST (FIG. 5G) and ALT (FIG. 14B), urea (FIG. 5H), and creatinine (FIG. 14C), and no severe toxicity to the liver or kidney was observed. Finally, a histologic examination of major organs was conducted via hematoxylin and eosin (H&E) staining (FIG. 5I). Comparable to the PBS group, there were no apparent morphological alterations or tissue damage observed in the mRNA / sLNP-treated group. These results underscored the biocompatibility and safety of the sLNP system.

[0049] EXAMPLES

[0050] All chemicals used for synthesis were purchased from MilliporeSigma, TCI, and Oakwood Chemical without further purification unless otherwise noted. MC3 (D- Lin-MC3-DMA) was purchased from MedChemExpress. DOPC (1 ,2-dioleoyl-sn- glycero-3-phosphocholine), DSPC (1 ,2-distearoyl-sn-glycero-3-phosphocholine), and DMG-PEG2k (1 ,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol) were purchased from Avanti Polar Lipids. Flash chromatography was performed on a puriFLASH® system (Advion).1H NMR spectra were collected on a Bruker 600 MHz Spectrometer. Mass spectra were recorded on an Advion Compact Mass Spectrometer. Firefly luciferase (fLuc) and Cre mRNA were purchased from TriLink Biotechnologies. In vivo and ex vivo bioluminescence signals were imaged using a Spectral Instruments Imaging AMI HTX system and Xenogen IVIS 50 system. Hydrodynamic size, polydispersity and zeta-potential were measured by a Malvern ZetaSizer Ultra. CryoEM measurements were performed on a JEOL JEM-21 OOF cryo transmission electron microscope. Confocal images were taken on a Leica SP8 confocal microscopy.

[0051] To synthesize DHSEH, hexadecyl sulfide was first prepared by reacting 1- hexadecanethiol with 1 -bromohexadecane. 2-Bromoethyl acetate was then reacted with hexadecyl sulfide in the presence of silver tetrafluoroborate, and the product was deprotected in HCI solution. DHSEH was then characterized by1H NMR and MS. DOSEH was synthesized using a similar route, except that octadecyl sulfide was synthesized in the first step using 1 -octadecanethiol and 1-boromooctadecane as reactants.

[0052] With respect to the fabrication and characterization of mRNA / sLNP, we prepared sLNP, sulfonium lipids (69.6%, weight ratio) were mixed with cholesterol (17.4%), DOPC (8.7%), and DMG-PEG2k (4.3%) in pure ethanol. It should be noted that the weight / molar percentages of these components can be varied from about 0.1 % to about 99.9% for each component. We further note that other formulations may also be used for the disclosed sulfonium lipids having components that range from about 0.1% to 100%, and other excipients may be included, as needed, in amount from about 0.1 % to about 99.9%.

[0053] Lipid nanoparticles were prepared by adding the lipid mix solution to sodium acetate buffer under vortex. The sLNP was then purified by dialysis against DI water (Slide-A-Lyzer™ dialysis cassette; ThermoFisher). To prepare mRNA / sLNP,precalculated mRNA and sLNP were mixed in nuclease-free water (sLNP / mRNA = 14 / 1 , w / w). After a brief pipette mixing, the solution was stored at room temperature for 20 min before use. A similar formulation process was used to prepare lung-tropic MC3-DOTAP LNPs. The organic phase contained 50% DOTAP (molar ratio), 25% MC3, 5% DSPC, 19.25% cholesterol, and 0.75% DMG-PEG2k. mRNA / MC3-DOTAP was prepared by mixing mRNA with MC3-DOTAP LNP in nuclease-free water at a weight ratio of 40 / 1 . Hydrodynamic size, polydispersity, and zeta-potential of mRNA / sLNP were measured by a Malvern ZetaSizer Ultra at room temperature. mRNA encapsulation efficacy (Wioaded mRNA / Wtotai mRNA* 100%) was measured by the Quant-it™ RiboGreen RNA Assay Kit (Thermo Fisher), following the manufacturer’s instruction. For cryoEM imaging, mRNA / sLNP were vitrified on Au-Flat 1 .2 / 1 .3 holey grids (Protochips) using a Leica EM GP2 cryo plunger set to 6 °C, 85% humidity, five seconds blot time. Grids were mounted on a Gatan 914 side entry cryo specimen holder and imaged in a JEOL JEM-21 OOF transmission electron microscope operating at 200 kV. Micrographs were recorded on a Gatan OneView CMOS camera at a magnification of x40,000 and a defocus of between -2.5 to -3.5 pm.

[0054] Our animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of SUNY Upstate Medical University. All studies were performed in accordance with the National Institutes of Health and ARRIVE guidelines for the use of laboratory animals. Balb / c, C57BL / 6, and Ai 14 mice (6-8-week-old; half male and half female) were purchased from the Jackson Laboratory. Caging systems and husbandry care were provided by the Department of Laboratory Animal Resources at Upstate Medical University.

[0055] For fLuc mRNA delivery and bioluminescence imaging, Balb / c mice were administered with PBS, free mRNA, mRNA / sLNP, or mRNA / MC3-DOTAP (0.1 mg mRNA / kg body weight) through retro-orbital injection. Six hours after these treatments, 100 pL D-luciferin in sterile PBS was injected intraperitoneally, animals were anesthetized with isoflurane, and bioluminescence images were recorded on a Spectral Instruments Imaging AmiHTX system or Xenogen IVIS 50 (for kinetics study), 10 min after D-luciferin injection. Mice were then sacrificed, and organs were collected for ex vivo imaging. The signal intensity was analyzed using AURA (AmiHTX) and Living Image (IVIS 50) software, respectively. The organ specificity was calculated as the signal intensity from a particular organ (e.g. lungs from mRNA / DHSEH-injected mice) subtracted by background signal (e.g. lungs from PBSgroup) and then divided by the total background-subtracted signal from all analyzed organs (i.e. liver, heart, spleen, lung, kidney, and brain).

[0056] For Cre mRNA delivery, Ai 14 mice were administered with PBS, Cre mRNA / sLNP, or Cre mRNA / MC3-DOTAP (0.42 mg mRNA / kg body weight) through retro-orbital injection. Five days post-injection, mice were sacrificed, and lungs were collected. For confocal imaging, lungs were embedded in OCT compound and sectioned into 10 pm slices using cryostat microtome (Leica CM 1950). Slices were washed with PBS three times, then stained with DAPI and imaged under a Leica SP8 confocal microscope. For flow cytometry analysis, lungs were digested into singlecell suspension using the Mouse Lung Dissociation Kit (Miltenyi Biotec), stained with FITC-labeled antibodies (anti-CDD31 for endothelial cells, anti-CD326 for epithelial cells, and anti-CD45 for immune cells; Invitrogen), and analyzed on a BD LSRII Cell Analyzer.

[0057] For proteomics analysis sLNP was added to adult C57BL / 6 mouse plasma (1 / 1 , v / v) and incubated for 60 min at 37 °C. The sLNP / plasma mixture was centrifuged at 13,000 xg and 4 °C for 30 min. The supernatant was removed and the pallet was washed with cold PBS three times. After resuspension, the protein concentration was measured by BCA assay (Pierce™ BCA Protein Assay Kit, Thermo Scientific). A total of 50 pg protein was used for proteomic analysis by the Upstate Medical University Proteomics Core.

[0058] In our inflammation and toxicity studies, Balb / c mice were administered with PBS or mRNA / DOSEH (0.1 mg mRNA / kg body weight) through retro-orbital injection. Body weight was recorded daily for five consecutive days. Mice were then sacrificed, and the bronchoalveolar lavage fluid was obtained from the mouse lung via lavage with three sets of 0.5 mL sterile saline washes. The collected fluid underwent centrifugation at 250 xg for 10 min, resulting in a pellet that was then suspended in 1 mL of sterile saline. For cell slide preparation, 200 pL of this suspension was centrifuged using a Cytospin centrifuge (Hettich ROTOFIX 32A) at 1000 rpm for 3 min, effectively depositing the cells onto a slide. The slide was air-dried and subsequently stained with Hema-3 (Fisher Scientific) for further analysis. Blinded reviewers quantified macrophages in the cell preparations by assessing 20 high- power fields (HPF) using a Nikon Eclipse TE2000-U microscope. The protein concentration in BALF was determined using the BCA assay. The levels of TNF-a, IL- 6, and IL-1 p in BALF samples were analyzed using ELISA kits (Invitrogen), followingthe manufacturer's instructions. Hematological analysis was conducted on a VetScan® HM5 Hematology Analyzer. Assay kits for AST, ALT, BUN, and creatinine were purchased from MilliporeSigma and used following the manufacturer's instructions. Tissue sectioning and H&E staining was performed in the Upstate Pathology Research Core. Three representative fields per slide were examined by blinded reviewers at x200 magnification under light microscopy.

[0059] Table 1 presents a summary of the top 20 most abundant proteins in the sLNP protein corona.

[0060] In our examples, we first synthesized sulfonium lipids (i.e. DHSEH and DOSEH), fabricated sLNPs through self-assembly, and demonstrated their application in mediating systemic delivery of mRNA to the lungs in adult mice. The sLNP- enabled systemic lung-specific mRNA delivery led to a uniform protein expression in all lung lobes, and it outperformed lung-tropic MC3-DOTAP formulation in terms of delivery efficacy and organ specificity. The kinetics study revealed that a higher dose induced greater and prolonged protein production. In the Ai14 mouse model, around 67.0% endothelial cells, 11.3% epithelial cells, and 3.5% immune cells in the lungs were transfected by mRNA / sLNP. Proteomic analysis of the sLNP protein corona shed light on the molecular mechanism of lung-targeting property, in which the fibrinogen, vitronectin, and / or AAT-mediated cell recognition and receptor-mediated internalization might be involved. The newly developed sLNP did not induce inflammation in the lung tissues, and no obvious toxicity to the lung, blood system, liver, kidney, heart, and spleen was observed. Collectively, these findings demonstrate that the sLNP can serve as a new class of carrier system for systemic delivery of mRNA to the lungs. We have demonstrated that non-conventional sulfonium lipid-based nanoparticles can efficiently complex with mRNA and facilitate in vivo delivery in an organ-specific manner. By leveraging the novel structure of sulfonium moiety, we successfully expanded the diversity of lipid chemistry available for designing lung-specific LNPs. Our sLNP formulations incorporate functional mRNA for therapeutic applications aimed at treating lung-associated diseases.REFERENCES

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[0062] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and / or structures for performing the function and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the inventive teachings is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0063] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0064] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0065] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0066] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0067] As used herein in the specification and in the claims, “or" should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims,“consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0068] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0069] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of” and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

We Claim:

1. A sulfonium lipid compound having a formula (R1)(R2)(R3)S+X-, wherein R1, R2, and R3 are organic groups, S+ is the positively charged sulfur atom, and X- is a negatively charged counterion.

2. The sulfonium lipid compound of claim 1 , wherein at least one of R1 , R2, and R3 groups contain at least one hydrophilic moiety and at least one of R1 , R2, and R3 groups contain at least one hydrophobic moiety.

3. The sulfonium lipid compound of claim 2, wherein hydrophilic moiety is at least one selected from the group consisting of hydroxyl, carboxyl, ether, amino, amide, amino acid, phosphonate, sulfate, sulfonate, carbohydrate, and carbonyl4. The sulfonium lipid compound of claim 2, wherein the hydrophobic moiety is at least one selected from the group consisting of linear or non-linear alkyl, phenyl, alkenyl, sterol, halogen, halogen ether, and oligo- or poly- ester, ether, amide, carbonate, urea, and urethane.

5. The sulfonium lipid compound of claim 1, wherein the negatively charged counterions is at least one selected from the group consisting of Chloride (Cl-), Bromide (Br-), Iodide (I-), Phosphate (PO4Λ3-), Carbonate (CO3A2-), Sulfate (SO4Λ2-) tetrafluoroborate (BF4-), trifluoromethanesulfonate (CF3SO3-), methanesulfonate (CH3SO3-), and tosylate (CH3C6H6SO3-).

6. The sulfonium lipid compound of claim 1, wherein the negatively charged counterions is DNA and / or RNA.

7. The sulfonium lipid compound of claim 1, wherein the compound is DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium), DOSEH ((2-hydroxyethyl)dioctadecyl sulfonium)), HOSEH (hexadevyl(2-hydroxyethyl)octadecyl sulfonium), DHSEA ((2- amino-2-oxoethyl)dihexadecyl sulfonium), or DOSPH ((3-hydroxypropyl)dioctadecyl sulfonium).

8. A composition for lung-specific mRNA delivery, comprising: a) a sulfonium lipid nanoparticie (sLNP); and b) an mRNA payload encapsulated within the sLNP.

9. The composition of claim 8, wherein the sulfonium lipid nanoparticie is at least one selected from the group consisting of DHSEH (Dihexadecyl(2-hydroxyethyl) sulfonium), DOSEH ((2-hydroxyethyl)dioctadecyl sulfonium)), HOSEH (hexadevyl(2- hydroxyethyl)octadecyl sulfonium), DHSEA ((2-amino-2-oxoethyl)dihexadecyl sulfonium), and DOSPH ((3-hydroxypropyl)dioctadecyl sulfonium).

10. The composition of claim 8, wherein the sLNP has a size range of about 50 nm to about 500 nm.

11. The composition of claim 8, wherein the sLNP has a positive zeta potential.

12. The composition of claim 8, wherein the sLNP comprises a mixture of sulfonium lipids, a helper lipid, sterol, and PEG-lipids.

13. The composition of claim 12, wherein the helper lipid is at least one selected from the group consisting of DSPC (distearoylphosphatidylcholine), DOPC (1,2- Dioleoyl-sn-glycero-3-phosphocholine), DMPC (1 ,2-Dimyristoyl-sn-glycero-3- phosphocholine), DPPC (1 ,2-Dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1 ,2- dioleoyl-sn-glycero-3-phosphoethanolamine), DOPC (1 ,2-dioleoyl-sn-glycero-3- phosphocholine), DOPS (1 ,2-dioleoyl-sn-glycero-3-phospho-L-serine), phosphatidylserine, and DOTAP (1,2-dioleoyl-3-trimethylammonium-propane).

14. The composition of claim 12, wherein the sterol is at least one selected from the group consisting of cholesterol, bile acid, β-Sitosterol, Stigmasterol, Fucosterol, Ergosterol, Fungisterol, 7-Dehydrocholesterol, Saringosterol.

15. The composition of claim 8, wherein the mRNA encapsulation efficacy is greater than 80%.

16. The composition of claim 8, wherein the sLNP demonstrates higher specificity for lung endothelial cells compared to other lung-targeting lipid nanoparticles.

17. A method for delivering mRNA to lung tissue, comprising: administering the composition of claim 8 intravenously to a subject in need thereof, wherein said method results in delivery of mRNA to the lungs without evidence of lung and systemic inflammation or toxicity in major organs and the mRNA is delivered to endothelial, epithelial, and immune cells, and wherein the pulmonary endothelial cells are primary targets.

18. A method of treating a lung-associated disease or condition, comprising: administering a therapeutically effective amount of the composition of claim 8 to a subject in need thereof.

19. A sulfonium lipid nanoparticle (sLNP) composition for delivering mRNA to the lungs, comprising: a) at least one sulfonium lipid; b) with or without one helper lipid; c) with or without sterol derivative; and d) with or without one PEG-lipid.

20. The method of claim 18, wherein the mRNA encodes a therapeutic protein for treating a lung-associated disease.

21. The composition of claim 16, wherein the mRNA encodes a therapeutic protein for treating a lung-associated disease.

22. A pharmaceutical composition for lung-specific mRNA delivery, comprising: a) an sLNP composition according to claim 1; b) a therapeutic mRNA; and c) a pharmaceutically acceptable carrier.

23. The pharmaceutical composition of claim 22, wherein the sLNP demonstrates lung-specific mRNA delivery following intravenous administration without evidence of lung inflammation or systemic toxicity.

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