Novel nebulized lipid nanoparticle compositions and uses thereof

Abstract

The present invention is based on the surprising discovery that a lipid nanoparticle (LNP) bearing sialic acid residues on the surface can withstand nebulization and maintain its integrity. The invention provides an LNP composition comprising a lipid bearing, or substituted by, a sialic acid (SA) residue such as a ganglioside, a ganglioside derivative, a mimetic, a SA-bearing entity or a combination thereof. The lipid nanoparticles described herein can incorporate nucleic acid molecules, or cargos, withstanding nebulization, and delivering the cargos to the lung via a nebulization-mediated delivery. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided herein are useful for the treatment of various pulmonary diseases. The LNPs provided herein are also useful for prophylactic applications including vaccines for infectious diseases, allergies, autoimmune diseases and cancer.

Description

[0001] NOVEL NEBULIZED LIPID NANOPARTICLE COMPOSITIONS AND USES THEREOF

[0002] RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 617,223, filed on January 3, 2024. The entire teachings of the above application are incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Recent advancements in in vivo delivery of nucleic acid therapeutics including messenger RNA (mRNA), small interfering RNA (siRNA), and antisense oligonucleotide (ASO), has brought hope to treat and prevent a variety of infectious, genetic, cancer and other diseases. Lipid nanoparticle (LNP)-based mRNA vaccines have demonstrated an excellent safety profile and efficacy against the SARS-CoV-2 virus during the COVID-19 pandemic. New therapeutic modalities, including cancer vaccines, protein replacement therapy, in vivo cell therapy, and genome editing are emerging and are in various development stages. Many of these new therapeutic modalities rely on viral delivery. However, viral delivery suffers from several shortcomings. For example, viral gene delivery systems exhibit limitations such as immunogenicity, mutagenesis, carcinogenesis, and low loading capacity. Therefore, more and more drug developers are exploring LNP systems for the delivery of their therapeutic cargos.

[0006] Delivery of nucleic acid cargos to the lung using LNP technology presents great opportunities to treat many lung diseases such as asthma, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), a-1 antitrypsin deficiency (AAT), pulmonary fibrosis and sarcoidosis, and lung cancer. In addition, delivery of vaccines to the deep lung may provide enhanced protection against respiratory infections compared with intramuscular or intranasal vaccines.

[0007] Currently, two strategies have been explored for the delivery of LNPs that incorporate RNA cargo to the lung. One of them is through systemic injection. This approach requires the LNP system to have a lung tropism - meaning that at least a portion of the delivered LNPs will accumulate in the lung. This systemic approach has not been very successful because conventional LNPs predominately accumulate in the liver, and for most of those “optimized” systems only a portion of the LNP delivered accumulates in the lung. A second approach is to directly deliver the LNPs to the lung through inhalation of nebulized LNPs. The advantages of the inhalation drug delivery system include reduced systemic toxicity and increased local drug concentration in the lungs. Therefore, inhalation is a preferred method for delivering therapeutics to the lung.

[0008] However, the inhalation approach must overcome several unique challenges. First, the current state-of-the-art LNPs cannot withstand the strong shear force generated even in the gentlest nebulizer. The nebulization process can disrupt LNP structure causing the encapsulation efficiency (EE) to dramatically decrease and therefore loss of the therapeutic cargo ensues. Nebulized LNPs also need to be able to penetrate through both cellular and extracellular barriers in the lung. Additionally, LNPs delivered to the lung may activate the complement cascade, cause lung thrombosis, and induce inflammation.

[0009] Therefore, there is an unmet need to develop a novel lipid nanoparticle system that is capable of delivering nucleic acid cargo, surviving nebulization without losing its integrity (e.g., size and EE), and penetrating through the lung mucus without causing thrombosis and inflammation.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention is based on the surprising discovery that a lipid nanoparticle (LNP) bearing sialic acid residues on the surface can withstand nebulization and maintain its integrity. The invention provides an LNP composition comprising a lipid bearing, or substituted by, a sialic acid (SA) residue such as a ganglioside, a ganglioside derivative, a mimetic, a SA-bearing entity or a combination thereof. The lipid nanoparticles described herein can incorporate nucleic acid molecules, or cargos, withstanding nebulization, and delivering the cargos to the lung via a nebulization-mediated delivery. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided herein are useful for the treatment of various pulmonary diseases. The LNPs provided herein are also useful for prophylactic applications including vaccines for infectious diseases, allergies, autoimmune diseases and cancer.

[0012] Sialic acid, also known as N-acetylneuraminic acid, is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectin (Siglec). Sialic acid has mainly three derivatives, N-acetyl neuraminic acid (Neu5 Ac or “NANA”), N-acetyl neuraminic acid hydroxyalkyl (Neu5Gc) and 3- deoxy-D-glycero-D-galacto-nonyl ketose (Kdn). There are other Siglec-binding sialic acid derivatives that are further derived from these primary derivatives.

[0013] Many Siglecs have an intracellular immunoreceptor tyrosine-based inhibition motif (ITIM) that can mediate inhibitory signals upon binding to sialic acid and activate downstream inhibitory signaling through the recruitment of tyrosine phosphatases SHP- 1 and SHP-2. Sialic acid can also regulate the alternative pathway of complement activation. Major serum protein complement factor H recognizes sialic acid as a “self’ marker, which helps to inhibit Clq / C3b fragment activation. Therefore, sialic acid, when binding a Siglec receptor on immune cells or the complement factor H (CFH), serves as a self-associated molecular pattern (SMAP) to suppress over-reactive immune responses and maintain an overall homeostasis.

[0014] To leverage the benefits of using sialic acid, one or more SA units can be chemically attached to a lipid, a polymer, a small molecule, or other chemical and biological entities to form “SA-bearing entities.” “SA-bearing” entities may be further incorporated into a lipid nanoparticle. Preferably, the sialic acid unit(s) are covalently bound to an end of a hydrophobic group, such as an optionally substituted C8-C20 alkyl or lipid group. Naturally occurring lipids, e.g. gangliosides, naturally contain SA units in their molecular structures.

[0015] Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked on the sugar chain. Preferred gangliosides include a ganglioside containing one sialic acid unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, or two sialic acid units such as GDla, GDlb, GD2 and GD3, or three sialic acid units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four sialic acid units such as GQ1. Other nonlimiting examples of gangliosides include ganglioside-total, C18:0(2-NBD) GM1, NGcGM3, C18:0 GM3, C20:0 GM1, C17:0 GM1 and C18:0 GM1, commercially available at Avanti Polar Lipids, Birmingham, AL.

[0016] For gangliosides that comprise only a single sialic acid unit (such as in the case of GM1, GM2 and GM3), the sialic acid unit can be linked with its neighboring sugar ring via an a2,3, a2,6, a2,8, or a2,9 linkage. For ganglioside that contains only one sialic acid unit, an a2,3 linkage is preferred. For gangliosides that comprise multiple sialic acid units, one sialic acid unit may be linked with its neighboring sialic acid unit via an a2,3, a2,6, a2,8, or a2,9 linkage. Typically, the lipid nanoparticles further comprise one or more chemical entities to form lipid nanoparticles of the present invention. Preferably the lipid nanoparticles further comprise one or more cholesterol or cholesterol derivatives, phospholipids, PEG-lipids, cationic lipids, and ionizable lipids.

[0017] A cationic lipid can be a natural or synthetic lipid. An example of a cationic lipid is an ammonium lipid, or lipid characterized by a positively charged nitrogen moiety. The cationic lipid can be substituted by a tertiary ammonium group, such as a trialkyl ammonium, preferably a trimethyl ammonium. The cationic lipid can be further substituted by one or more substituted or unsubstituted long chain alkyls or alkenyls, such as a C4-C20 alkyl or alkenyl. Examples of commonly used lipids include multivalent cationic lipid, DOTMA, ethyl PC’s, DDAB, pH sensitive lipids, dioleoyl-3- trimethylammonium propane (DOTAP), DC-cholesterol, and GL67.

[0018] An ionizable lipid can be neutral and non-ionic at physiological pH but will be protonated to become positively charged at lower pHs. Examples of commercially available ionizable lipids include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, ALC-0315, SM- 102, LP-01, DODMA, and DODAP.

[0019] PEG-lipids include Poly(ethylene glycol) derivatives that are attached to a lipid moiety such as DMG or DSPE. The number of ethylene glycol (EG) units in the PEG-lipid can preferably be three (3) or more. For example, PEG(IOOO) represents a PEG unit having a MW of 1000, which is equivalent to about 22-23 EG units. PEG-lipids that are commonly used in making lipid nanoparticles include DSPE-PEG(IOOO), DSPE-PEG(2000), DSPE- PEG(5000), DMG-PEG(IOOO), DMG-PEG(2000), DMG-PEG(5000), Bis-DSPE PEG2000, and derivatives thereof. PEG-lipid can also be functionalized for bioconjugation.

[0020] In one particular embodiment, the LNP of the present invention comprises a ganglioside, an ionizable lipid, cholesterol or a cholesterol derivative, a phospholipid, and a nucleic acid cargo. It is to be noted that the use of PEG-lipid is not required for the fabrication of the LNP of the present invention. However, a PEG-lipid may be added to the composition of the LNP of the present invention for the adjustment of its overall performance properties.

[0021] Specifically, the LNP of the present invention can be nebulized to form aerosolized LNPs that substantially retain their original particle size and encapsulation efficiency (EE); for example, the average particle size (Z-average) of the LNP after the nebulization is preferably between 90% to 125%, such as 100%, 105%, 110%, 115%, 120%, or 125%, of the original Z-average particle size measured before the nebulization process; and the LNP retains between 75% and 100%, such as 100%, 95%, 90%, 85% or 80%, of its original EE before the nebulization process.

[0022] The invention further relates to methods for the treatment of a disease or disorder in the lung, such as asthma, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), a-1 antitrypsin deficiency (AAT), cystic fibrosis (CF), pulmonary fibrosis, pulmonary sarcoidosis, lung cancer, an inflammatory disease, or an autoimmune disease, in a subject in need thereof comprising administering to the subject the composition of the invention.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 A and IB provide particle size and encapsulation efficiency results for formulations described below.

[0025] FIG. 2A and 2B show the number of transfected endothelial cells per square pm in the lung tissue collected from mice having inhaled nebulized Cytofinity LNPs.

[0026] FIG. 2C shows the number of transfected ciliated cells per square pm in the lung tissue collected from mice having inhaled nebulized Cytofinity LNPs.

[0027] DETAILED DESCRIPTION OF THE INVENTION

[0028] Definitions

[0029] As used herein, “pharmaceutically acceptable” includes those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for medical or veterinary use when in contact with the tissues of human beings and animals at the concentration, dosage or amount present in the product, without causing excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Preferably, a pharmaceutically acceptable material (e.g., polymer, excipient, surfactant, solvent, or microparticles / nanoparticles produced therefrom) is suitable or approved for human medical use.

[0030] As used herein, “nanoparticles” are preferably roughly round, sphere, or sphere-like in shape, and are generally within the size range of, e.g., between about 1-1,000 nm, between about 10-1,000 nm, or between about 50-1,000 nm, or between about 100-500 nm, as measured by laser diffraction, for example. The subject nanoparticles may also include particles that are less likely to clump in vivo. Particle size and size distribution can be measured by a dynamic light scattering instrument, e.g., a Malvern Zetasizer. The particle size is typically reported as Z-average mean diameter. Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation. The term “nanoparticle” is not intended to convey any specific shape limitation. Such particles include, but are not limited to, those having a generally polyhedral or spherical geometry. Preferred particles are characterized by a spherical geometry typically produced by emulsion-based encapsulation processes. It is understood that the terms “microparticle” and “nanoparticle” are used interchangeably herein, unless accompanied by a specific description of size. For example, the term “microparticles” is intended to also embrace “nanoparticles” as if stated as “microparticles and / or nanoparticles” unless the context demands otherwise.

[0031] The term “particle” encompasses both nanoparticle and microparticles.

[0032] As used herein “a” or “an” means one or more unless otherwise specified.

[0033] As used herein, “about” generally means up to ±10% of the particular term being modified.

[0034] The terms “sialic acid residue” and “sialic acid moiety” as well as their plural referents, and the like, are used interchangeably herein.

[0035] As used herein, the term “subject” is used to mean an animal, preferably a mammal, including a human or non-human. The terms “patient” and “subject” may be used herein interchangeably.

[0036] “Treatment” or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing own or preventing the onset, progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease. As used herein, “treatment” (and grammatical variations thereof such as “treat” or “treating”) includes to clinical intervention to alter the natural course of a disease in the individual being treated and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, combinations of the invention are used to delay development of a disease or to slow the progression of a disease.

[0037] Ganglioside Gangliosides are molecules composed of glycosphingolipid with one or more sialic acids linked on the sugar chain. They form lipid rafts in the outer leaflet of the cell plasma membrane, especially in neuronal cells in the central nervous system. Gangliosides have been found to be highly important molecules in immunology as they participate in cellular proliferation, differentiation, adhesion, signal transduction, cell-to-cell interactions, tumorigenesis and metastasis. More than 60 gangliosides are known.

[0038] Ganglioside GM2

[0039] Ganglioside GQ1

[0040] Gangliosides can be named based on the number of sialic acid (SA) units they have in the molecule. Thus, gangliosides having one SA unit are named as “GM” such as GM1, GM2 and GM3. Here, “G” stands for “ganglioside,” and “M” stands for “mono.” Similarly, “GD”, “GT” and “GQ” would refer to gangliosides having two (“di”), three (“tri”), and four (“quadruple”), respectively. As an illustration, the structures of several representative gangliosides are shown above.

[0041] The SA unit in the ganglioside molecules can bind Siglecs expressed on various types of cells including immune cells. Major serum protein complement factor H (CFH) recognizes sialic acid as a “self’ marker, which helps to inhibit Clq / C3b fragment activation. Gangliosides can also bind CFH of the complement system. When binding Siglecs on immune cells and / or the complement system, ganglioside serves as a “Self- Associated Molecular Pattern” or SAMP to mitigate the inflammation. Thus, lipid nanoparticles incorporating a ganglioside molecule on the nanoparticle surface can provide high avidity and efficiency for binding Siglecs to resolve or mitigate inflammation. The ganglioside may be a ganglioside containing one SA unit such as GM1, GM2, GM3, asialo-GMl, GAI, asialo- GM2, GA2, or two SA units such as GDI a, GDlb, GD2 and GD3, or three SA units such as GTla, GTlb, GTlc, OAc-GTlb, GT3, or four SA units such as GQ1. The ganglioside preferably has 1, 2, 3, 4 or more SA units. In one embodiment, the ganglioside is selected from the group containing GM1, GM3, and GD3.

[0042] Other non-limiting examples of gangliosides include ganglioside-total, Cl 8:0 (2- NBD) GM1, NGcGM3, C18:0 GM3, C20:0 GM1, C17:0 GM1 and C18:0 GM1; commercially available at Avanti Polar Lipids, Birmingham, AL. For gangliosides that comprise only a single SA unit (such as in the case of GM1, GM2 and GM3), the SA may be linked with its neighboring sugar ring via an a2,3, a2,6, a2,8, or a2,9 linkage. Gangliosides that contain an a2,3 linkage are preferred.

[0043] The gangliosides can be added to the formulation in an amount of at least about 0.1% weight percentage of total solids in the nanoparticle composition. The ganglioside can preferably be between 5-85%, 10-75%, or, more preferably between 20 and 40% of the total lipid composition. Alternatively or additionally, the molar ratio of the ganglioside to total lipids can be between 1-50%, 2-20%, 3-15%, 5-15% or about 10%.

[0044] Cholesterol

[0045] The lipid nanoparticle described herein preferably further comprises cholesterol or a cholesterol derivative. Cholesterol can be natural cholesterol in its native form or synthetic derivatives. Natural cholesterol has a chemical structure shown below. Cholesterol derivatives include compounds comprising the four cyclic ring system of cholesterol, preferably a compound characterized by the formula: wherein Ri is selected from hydrogen or a substituted or unsubstituted alkyl, such as a Cl to C4 alkyl; R2 is selected from a substituted or unsubstituted, saturated or unsaturated alkyl, such as a Cl to C12 alkyl or alkenyl; and the dashed line can be a single or double bond.

[0046] Cholesterol derivatives also include vitamin D and other open ring derivatives including cholecalciferol and ergocalciferol: and compounds having the structure:

[0047] wherein Ri is selected from hydrogen or a substituted or unsubstituted alkyl, such as a Cl to C4 alkyl; R2 is selected from a substituted or unsubstituted, saturated or unsaturated alkyl, such as a Cl to C12 alkyl or alkenyl; R3 is hydrogen, methyl or methylene, and each dashed line is independently a single or double bond.

[0048] The cholesterol or cholesterol derivative can be added to the formulation in an amount of at least about 0.1% weight percentage of total solids in the nanoparticle composition. The molar ratio of the cholesterol or cholesterol derivative can preferably be between 1-50%, 10-45%, or, more preferably between 15 and 40% of the total lipid composition.

[0049] Phospholipid

[0050] The lipid nanoparticles preferably further comprise one or more phospholipids.

[0051] Phospholipids are a class of lipids that have in their molecular structures a hydrophilic "head" containing a phosphate group and one, two or more hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (such as a glycerol molecule). The phosphate group can be further substituted with various chemical moieties. Examples of such chemical moieties include serine, ethanolamine, choline, glycerol, inositol, and polyethylene glycol (PEG). Phosphatidylcholines are preferred.

[0052] Phospholipids include but are not limited to distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-0-monom ethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, l-stearoyl-2-oleoyl-phosphatidy ethanol amine (SOPE), l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (trans DOPE), phospholipid-PEG, the derivatives and combinations thereof.

[0053] In a preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0054] The phospholipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the phospholipid can preferably between 1-50%, 2-20%, 3-15%, 5-15% or about 10% of the total lipid composition.

[0055] Cationic and Ionizable Lipid

[0056] In a particular embodiment, the anti-inflammatory lipid nanoparticle further comprises one or more cationic or ionizable lipids. A cationic lipid is a lipid having a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge. Preferably, the cationic lipid can precipitate with the nucleic acid molecule or cargo.

[0057] In some embodiments, the cationic lipid can be selected from, for example Dioleoyl-3- trimethylammonium propane (DOTAP), l,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(didodecylamino)-Nl,Nl,4-tridodecyl-l- piperazineethanamine (KL 10), N1 -[2-(didodecylamino)ethyl]-N 1 ,N4,N4-tridodecyl- 1 ,4- piperazinediethanami- ne (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy- N,N-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]- dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)- [1,3] -di oxolane (DLin-KC2-DMA), 1,2- dioleyloxy-N,N- dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3- yl oxy] octyl} oxy )-N,N- dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l-yl oxy]propan-l -amine (Octyl-CLinDMA), (2R)-2-({8-[(3.beta.)- cholest-5-en-3-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9- Z,12Z)- octadeca-9,12-dien-l-yloxy]propan-l -amine (Octyl-CLinDMA (2R)), and (2S)-2-({8- [(3.beta.)-cholest-5-en-3- yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z- ,12Z)-octadeca-9,12- dien-1- yloxy]propan-l- amine (Octyl-CLinDMA (2S)).

[0058] An ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH but will be protonated to become positively charged at lower pHs. Examples of commercially available ionizable lipids include DLin- KC2-DMA, DLin-MC3- DMA, DLin-DMA, DODMA, DODAP, ALC- 0315, LP-01, SM-102, SS-OP, SS-EC, etc.

[0059] In a preferred embodiment, the cationic or ionizable lipid is ALC-0315, which has the structure shown below, where the nitrogen can be protonated:

[0060] The ionizable lipid can be added to the formulation in an amount of at least about 0.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the cationic or ionizable lipid can preferably between 1-60%, 10-55%, or, more preferably between 20 and 50% of the total lipid composition.

[0061] PEG lipid

[0062] Polyethylene Glycol (PEG) lipids can also be used. The term "PEG lipid" refers to polyethylene glycol (PEG)-modified lipids. Non-limiting examples of PEG lipids include PEG- modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG- modified 1,2- diacyloxypropan-3-amines. Such lipids are also referred to as PEGylated lipids. In some embodiments, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG- DPPC, or a PEG-DSPE lipid. It is to be noted that in the current invention, the addition of PEG-lipid is optional. It is beneficial to construct the LNPs without a PEG-lipid as PEG-lipid may lead to the formation of anti-PEG antibodies, which may cause anaphylaxis, accelerated blood clearance, and unwanted immunogenicity. The ganglioside in the lipid nanoparticles of the current invention can act to stabilize the nanoparticles without the presence of PEG. The sialic acid residues on the LNPs also serve as SAMP to mitigate inflammatory reactions and maintain homeostasis. Active Agent

[0063] The lipid nanoparticle described herein can optionally further comprise an active agent, such as an anionic active agent, such as a nucleic acid molecule, or cargo. The nucleic acid cargo can be encapsulated within said lipid nanoparticle. The amount of the active agent can be about 0.01 to about 50% (w / w) of the nanoparticle total solids, or about 0.05 to about 25%, about 0.1 to about 10%, about 0.2 to about 5%, about 0.5 to about 3%, about 1 to about 5%, or about 2 to about 5% (w / w) of the nanoparticle total solids. The weight ratio of lipids to nucleic acid can be about 1 to 20, preferably 3 to 15, more preferably 4 to 10.

[0064] The concentration of nucleic acids to LNPs can be characterized by a N:P ratio. For example, N refers to the number of nitrogen atoms in the ionizable or cationic lipid (typically 1 nitrogen per molecule). P refers to the number of phosphates in the nucleic acid molecule. The nucleic acid molecule can be added to the lipid nanoparticle in a ratio preferably between 1 :2 to 10: 1, such as between 1 : 1 to 10: 1, more preferably between 3: 1 to 8: 1, such as 6: 1 or 8: 1.

[0065] In certain aspects, the active agent is advantageously an anionic drug (also referred to herein as an active pharmaceutical ingredient, or API). However, active agents that are non-therapeutic, such as diagnostics, can also be included as part of the particles according to the methods. Preferred active ingredients are oligonucleotides, nucleic acid molecules and mimics thereof, such as DNA, RNA, PNA, siRNA, microRNA, circular RNA, antisense, oligonucleotide, aptamer, and a combination thereof. The term “API” and “cargo” are used interchangeably herein.

[0066] Production of the Particles

[0067] The lipid nanoparticle described herein can be manufactured by a coprecipitation process. In general, the ganglioside can be dissolved along with other lipids (such as ionizable lipid, DSPC and cholesterol) in a preferably water-miscible organic solvent, such as an alcohol or ethanol. The alcohol solution containing the ganglioside and other lipids is combined slowly (e.g., dropwise) or with mixing (e.g., via a microfluidic device) with an aqueous buffer solution containing the RNA cargo to incur nanoprecipitation. For example, a small amount of organic solution can be added to the aqueous phase with mixing.

[0068] Preferably, the particles are manufactured using a pre-assembled device such as a T- mixer, or an automated, microfluidic device such as NanoAssemblr of Precision Nanosystems, Automated Nanoparticle System of Particle Works, and iNano L+ of Micro&Nano Biologies.

[0069] Exemplary solvents miscible with water include methanol, ethanol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0070] Solvent is then removed and / or particles collected, for example, by evaporation, solvent exchange, centrifugation or filtration, dialysis, tangential flow filtration, followed by dehydration, e.g., concentration or lyophilization.

[0071] The aqueous solution can optionally comprise a surfactant comprising organic or inorganic pharmaceutical excipients; various polymers; oligomers; natural products; nonionic, cationic, zwitterionic, or ionic surfactants; and mixtures thereof. The surfactant may comprise an acidic buffer such as citrate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), a polysorbate (Tween series) surfactant, a PEO- PPO-PEO (polyethylene oxide-polypropylene oxide-polyethylene oxide) triblock copolymer (Pluronic series or Poloxamer series) surfactant, or a t-octylphenyl-polyethylene glycol (Triton X- 100) surfactant or a salt, derivative, copolymer, or mixture thereof.

[0072] Such obtained nanoparticle suspension (either by co-precipitation or with a microfluidic system) can be further purified with dialysis, centrifugation, or tangential flow.

[0073] Particle Sizes

[0074] The size of the subject nanoparticles is from about 1 nm to about 10 pm, preferably from about 10 nm to about 2 pm, and more preferably from about 20 nm to about 1 pm, and most preferably from about 40 nm to about 500 nm. For example, nanoparticles may have average sizes between about 50 and 900 nm, such as about 50, 75, 100, 300, 500, 700, or 900 nm.

[0075] As used herein, particle size can be determined by any conventional particle size measuring techniques well known to those skilled in the art. Such techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation.

[0076] Pharmaceutical Composition

[0077] One aspect of the present invention provides pharmaceutical compositions which comprise the subject lipid nanoparticles, and optionally comprise a pharmaceutically acceptable carrier or excipient. Preferably, these compositions optionally further comprise one or more additional therapeutic agents. Alternatively, the subject particles of the current invention may be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents. For example, additional therapeutic agents for conjoint administration may be an approved anti-inflammatory agent, an immunotherapeutic agent, or a chemotherapeutic agent, or it may be any one of a number of agents undergoing approval in the Food and Drug Administration.

[0078] Preferably, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, preservatives, lubricants and the like, as suited to the particular dosage form desired. Remington ’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, PA, 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional carrier medium is incompatible with the compounds of the invention, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

[0079] Aerosols

[0080] Methods of delivering LNPs by aerosol inhalation are provided. An aerosol, such as a mist, can be delivered using air, oxygen, and / or oxygen and mixtures as a carrier gas. The aerosol can be delivered at room temperature or heated. The LNPs can be delivered to a fluid lining of the alveolar region of the lungs.

[0081] Devices used to deliver LNPs as aerosols can be based on, for example, nebulizers, pressurized metered-dose inhaler (pMDI), and dry powder inhalers (DPIs). Devices for inhaled drug delivery have two basic functions, namely aerosol formation and facilitation of aerosol transport into the lungs. A distinction is made between passive and active devices. A passive device derives the energy required for aerosol formation from the inhaled air stream, i.e., from the patient, while active devices create the aerosol independently of the patient's inhalation. Inhalation devices can be further categorized in various ways, such as single-dose versus multi-dose, or disposable versus reusable. Multi-dose devices can provide benefits for chronic therapy, such as cost reduction, portability, and portability, ease of use and convenience. For irregular administrations and one-time applications, disposable devices may be more suitable. Furthermore, aspects such as the risk of device contamination acting as a reservoir for microbial growth and allowing the development of antibiotic resistance may affect the choice for a multi- or single-dose device. Inhalation devices that can be used for pulmonary delivery: pressurized metered dose inhalers (pMDIs), nebulizers, soft mist inhalers, and dry powder inhalers (DPIs).

[0082] In general, pMDIs generate aerosol faster than the patient can inhale. Coordination between device actuation and patient inhalation is especially difficult in children and the elderly. With some DPIs, it is required that the patient inhales at maximum force to disperse then inhale the powder, which unless properly trained, is rarely achieved. In these scenarios, most of the aerosol deposits in the upper airways. For pMDIs, this problem can be addressed by providing a spacer or by designing a breath-activated inhaler instead of breath-coordinated devices.

[0083] Jet and ultrasonic nebulizers differ in the force used to generate an aerosol from the respective liquid. Depending on the type, nebulizers can generate 1-5 pm droplets. Nebulizers do not require patient coordination between inhalation and actuation, thus they are useful for pediatric, elderly, ventilated, non-conscious patients, or those who are unable to use pMDIs or DPIs. Nebulizers have the capability of delivering larger doses compared to the other aerosol devices even though this will require longer administration times.

[0084] Nebulizer devices can be breath-enhanced, breath-actuated, and vibrating mesh nebulizers. The design of breath-enhanced jet nebulizers is modified to allow for air entrainment during inspiration and to vent the expired air outside of the device. The main advantage of this approach is to increase the output rate, which in turn will decrease the administration time.

[0085] Breath-actuated nebulizers emit aerosolized droplets only when the patient inhales. Therefore, no drug is wasted during exhalation as the case of regular jet nebulizers and dissemination of expensive or toxic drugs to the surrounding environment is avoided.

[0086] Vibrating mesh nebulizers have a mesh plate that, when it vibrates through the action of the piezoelectric element, breaks the liquid into very fine droplets, which increases the volume of aerosol deposited in the alveoli. Vibrating mesh nebulizers can have an electronic indicator that show when the patient is breathing appropriately and only then, it releases the dose, with droplet size of mass median aerodynamic diameter, 4 pm and minimum drug loss (~1%). Smart devices can include a vibrating mesh nebulizer coupled with an adaptive aerosol delivery software that adjusts the aerosol emission based on the breathing pattern of the patient, which reduces drug loss and increases the inhaled mass. Such a device can adjust the dose delivery based on patient's last three breaths and provide feedback after dose delivery.

[0087] Soft Mist Inhaler (SMI) inhalation devices can be used to deliver the compositions described herein. The SMI is a nebulizer, as it disperses a solution of the active agent into fine droplets. It differs from a traditional nebulizer in that it is a hand-held, portable device that does not require an external power source, but is actuated by a mechanical spring. The instantaneous formation of the aerosol is comparable to a pMDI; thus, proper actuationinhalation coordination is necessary. While it generally takes longer before the entire aerosol is generated (1.5 s versus 0.21-0.36 s for an HFA-pMDI) and the aerosol is emitted as a slow- moving mist, this allows for a relatively high lung deposition.

[0088] Devices include, for example, continuous mode nebulizers Flo-Mist (Phillips) and Hope (B&B Medical Technologies) and the accessories such as regulators, e.g., Medipure™ Heliox-LCQ System (PraxAir) and control box, e.g., Precision Control Flow (PraxAir).

[0089] As described above, there is an unmet need to deliver nucleic acid therapeutics to the lung via inhalation, but traditional LNPs cannot withstand the strong shear force generated even in the gentlest nebulizer. The nebulization process can cause LNPs to form aggregates and increase their particle sizes. The shear force during nebulization may also disrupt the LNP structure, causing the encapsulation efficiency (EE) to dramatically decrease, and therefore, the loss of the therapeutic cargo ensues.

[0090] The present invention is based on the surprising discovery that the LNP comprising a ganglioside replacing some or all of the PEG-lipid in the LNP formulation can withstand nebulization and maintain its integrity. Specifically, the LNP of the present invention can be nebulized to form aerosolized LNPs that substantially retain their original particle size and encapsulation efficiency (EE); for example, the average particle size (Z-average) of the LNP after the nebulization is preferably between 90% and 125%, such as 100%, 105%, 110%, 115%, 120%, or 125%, of the original Z-average particle size measured before the nebulization process; and the LNP retains between 80% and 100%, such as 100%, 95%, 90%, 85% or 80%, of its original EE before the nebulization process.

[0091] Without being bound by any theory, the current inventor believes that the relatively long molecular chains of the PEG units on the surface of traditional LNPs may cause interparticle entanglements under the strong shear forces generated during the nebulization process. Such inter-particle entanglements can disintegrate the LNPs. By replacing these PEG units with ganglioside molecules, such entanglements can be prevented. Thus, the LNP described herein can be safely nebulized and effectively delivered to a subject's lung via inhalation.

[0092] Various types of personal vaporizers can be used to deliver the therapeutic compositions described herein and are known in the art. In general, personal vaporizers are characterized by heating a solid drug or compound. Vaporizers can work by directly heating a solid drug or compound to a smoldering point. Vaporizing a solid or solid concentrate can be done by convection on conduction. Convection heating of solid concentrate involves a heating element coming into contact with water, or another liquid, which then vaporizes. The hot vapor in turn directly heats the solid or solid concentrate to a smoldering point, releasing a vapor to be inhaled by a user. Conduction heating involves direct contact between the solid or solid concentrate and the heating element, which brings the solid to a smoldering point, releasing vapor to be inhaled by a user. Though vaporizers present advantages over smoking in terms of lung damage, the drug / active agent that is vaporized can be substantially deteriorated by the vaporizing heat.

[0093] Exemplary Uses

[0094] The lipid nanoparticles and compositions thereof have numerous applications including therapeutic methods.

[0095] Preferably, the nanoparticles or the composition comprising the nanoparticles can be used in a method of treating a disease or condition in a subject in need thereof, or a method of reducing the duration or severity of the disease or condition in the subject in need thereof, wherein the disease or condition is treatable with the particles (and optionally with a specific API), comprising administering a composition or a pharmaceutical composition comprising the particles to the subject, thereby treating the disease or condition. Where the particles comprise (for example, encapsulate) an API, the particles can be used in a method of administering or delivering the API to a subject in need thereof and / or for a method of treating a subject suffering from a disease or condition that can be treated with the API. For example, when the API is an anti-cancer agent, the particles can be administered to a subject with a cancerous condition.

[0096] The lipid nanoparticles described herein can be used to treat an inflammatory condition. Examples of such diseases and conditions include, but are not limited to, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, celiac disease, hyper-IgM immunodeficiency, arteriosclerosis, atherosclerosis, coronary artery disease, sepsis, myocarditis, encephalitis, transplant rejection, hepatitis, thyroiditis (e.g. Hashimoto's thyroiditis, Graves disease), osteoporosis, polymyositis, dermatomyositis, Type I diabetes, Type II diabetes, gout, dermatitis, alopecia areata, systemic lupus erythematosus, Sjogren's syndrome, lichen sclerosis, scleroderma, ulcerative colitis, diabetic retinopathy, pelvic inflammatory disease, periodontal disease, arthritis, juvenile chronic arthritis (e.g. chronic iridocyclitis), psoriasis, osteoporosis, nephropathy in diabetes mellitus, asthma, pelvic inflammatory disease, chronic inflammatory liver disease, chronic inflammatory lung disease, lung fibrosis, liver fibrosis, chronic inflammatory lung disease, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, peritonitis, cardiovascular disease, reperfusion injury, ischemia injury, stroke, burns, and other acute and chronic inflammatory diseases of the Central Nervous System (CNS; e.g. multiple sclerosis), gastrointestinal system, the skin and associated structures, the immune system, the hepato-biliary system, or any site in the body where pathology can occur with an inflammatory component. Inflammatory diseases also include diseases involving the gastrointestinal tract and associated tissues (such as ileus, appendicitis, peptic, gastric and duodenal ulcers, peritonitis, pancreatitis, ulcerative, pseudomembranous, acute and ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, coeliac disease, hepatitis, Crohn's disease, enteritis, and Whipple's disease); systemic or local inflammatory diseases and conditions (such as asthma, anaphylactic shock, immune complex disease, organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, and sarcoidosis); diseases involving the urogenital system and associated tissues (such as septic abortion, epididymitis, vaginitis, prostatitis, and urethritis); diseases involving the respiratory system and associated tissues (such as bronchitis, emphysema, rhinitis, cystic fibrosis, pneumonitis, adult respiratory distress syndrome, pneumoultramicroscopicsilicovolcanoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, and sinusitis); diseases arising from infection by various viruses (such as influenza, respiratory syncytial virus, HIV, hepatitis B virus, hepatitis C virus and herpes), bacteria (such as disseminated bacteremia, Dengue fever), fungi (such as candidiasis) and protozoa! And parasites multicellular (such as malaria, filariasis, amebiasis, and hydatid cysts); dermatological diseases and conditions of the skin (such as burns, dermatitis, dermatomyositis, sunburn, urticaria warts, and wheals); diseases involving the cardiovascular system and associated tissues (such as stenosis, restenosis, vasculitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, congestive heart failure, myocarditis, autoimmune myocarditis, myocardial ischemia, periarteritis nodosa, and rheumatic fever); diseases involving the central or peripheral nervous system and associated tissues (such as Alzheimer's disease, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, and uveitis); diseases of the bones, joints, muscles and connective tissues (such as the various arthritides and arthralgias, osteomyelitis, fasciitis, Paget's disease, gout, periodontal disease, rheumatoid arthritis, and synovitis); other autoimmune and inflammatory disorders (such as myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcets's syndrome, allograft rejection, graft-versus-host disease, Type I diabetes, ankylosing spondylitis, Berger's disease, and Retier's syndrome); as well as various cancers, tumors and proliferative disorders (such as Hodgkins disease); and, in any case the inflammatory or immune host response to any primary disease.

[0097] Diseases that can be treated also include allergic disorders or conditions, including allergic disease, allergy, eczema, asthma, allergic rhinitis or skin hypersensitivity. The disease to be treated can also be a viral infection, including, for example, a hepatitis virus infection, a West Nile virus infection, a flavivirus, an influenza infection, a rhinovirus infection, a papillomavirus infection, a paramyxovirus infection, or a parainfluenza virus infection. Preferably, the viral infection infects the central nervous system of said subject. Preferably, the viral infection causes viral encephalitis or viral meningitis. In yet another aspect, the disease to be treated is a bacterial infection. Exemplary bacterial infections are staphylococcus infections, streptococcus infections, mycobacterial infections, bacillus infections, Salmonella infections, Vibrio infections, spirochete infections, and Neisseria infections. Preferred are bacteria that infect the central nervous system of the subject. Most preferred are bacteria that cause encephalitis or meningitis.

[0098] In certain embodiments, the subject is a human patient. In certain embodiments, the subject is a non-human mammal, such as a non-human primate, a livestock animal (horse, mule, cattle, bull, cow, sheep, goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.), or a pet (cat, dog).

[0099] EXEMPLIFICATION

[0100] Preparation of lipid nanoparticles capable of withstanding nebulization

[0101] Example A: Preparation of an LNP comprising ALC-0315, Choi, DSPC, and GD3 in a molar ratio of 46:35:9:10

[0102] Stock solutions of the ionizable lipid ALC-0315, cholesterol (Choi), and 1,2- distearoyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A ganglioside GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. The lipid packet was made with 1.2x excess by combining 38.1 pL ALC-0315 solution, 14.6 pL Choi solution, 7.67 pL DSPC solution, and 32.52 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in 1.07x excess) for a 6:1 N:P ratio, 50 pL of the DasherGFP® mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0103] The lipid and cargo packets were loaded into a 10-mL Luer-lock syringes which were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: total flow rate (TFR) = 32 mL / min; cargolipid flow rate ratio (FRR) = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Suspended nanoparticles (4 mL) were collected from the T-mixer in a 50 mL conical tube and diluted by adding 46 mL phosphate buffered saline (PBS). To remove alcohol, the LNP suspension was transferred into a pre-hydrated Slide- A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS overnight.

[0104] Example B: Preparation of an LNP comprising MC3, Choi, DSPC, and GD3 in a molar ratio of 41:39:10:10

[0105] 20 mg / mL stock solutions of the ionizable lipid DLin-MC3-DMA (MC3), Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A ganglioside GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving appropriate amount of solid GD3 in methanol. The lipid packet was made with 1.2x excess by combining 31.87 pL MC3 solution, 18.25 pL Choi solution, 9.57 pL DSPC solution, and 36.48 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in 1.07x excess) for a 6:1 N:P ratio, 50 uL of the DasherGFP® mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0106] The lipid and cargo packets were loaded into a 10-mL Luer-lock syringes which were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T-mixer settings, controlled by KDS Legato software, were as follows: TFR = 32 mL / min; FRR = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Suspended nanoparticles (4 mL) were collected from the T-mixer in a 50 mL conical tube and diluted by adding 46 mL PBS. To remove alcohol, the LNP suspension was transferred into a prehydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed overnight against PBS.

[0107] Example C: Preparation of an LNP comprising MC3, Choi, DSPC, and GD3 in a molar ratio of 46:35:9:10

[0108] Stock solutions of the ionizable lipid MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A ganglioside GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. The lipid packet was made with 1.2x excess by combining 31.87 L MC3 solution, 14.6 L Choi solution, 7.67 pL DSPC solution, and 32.52 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in 1.07x excess) for a 6: 1 N:P ratio, 50 pL of the DasherGFP® mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0109] The lipid and cargo packets were loaded into 1 mL and 3 mL syringes, respectively. These were mounted on a microfluidic mixing instrument and secured to the inlet ports of a single-use cartridge. The flow rate was set at 15 ml / min for the RNA packet and 5 ml / min for the lipid solution, for a TFR of 20 mL / min and FRR of 3 : 1. Suspended nanoparticles (4 mL) were collected and diluted by adding 46 mL PBS. To remove alcohol, the LNP suspension was transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed overnight against PBS. The particles were concentrated for 10 minutes at 2000 RCF in a Vivaspin™ ultrafiltration spin column with a 100K MWCO. The sample was collected from the upper filter and stored at -80° C in a 5% w / v sucrose solution.

[0110] Example D: Preparation of an LNP comprising MC3, Choi, DSPC, and GD3 in a molar ratio of 49:37:9:5

[0111] Stock solutions at 20 mg / mL concentrations of the ionizable lipid MC3, Choi, and DSPC were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A ganglioside GD3 stock solution at a concentration of 10 mg / mL was prepared by dissolving an appropriate amount of solid GD3 in methanol. The lipid packet was made with 1.2x excess by combining 31.87 pL MC3 solution, 14.49 pL Choi solution, 7.2 pL DSPC solution, and 15.26 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in excess) for a 6: 1 N:P ratio, 50 pL of the DasherGFP® mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0112] The lipid and cargo packets were loaded into 1 mL and 3 mL syringes, respectively. These were mounted on a microfluidic mixing instrument and secured to the inlet ports of a single-use cartridge. The flow rate was set at 15 ml / min for the RNA packet and 5 ml / min for the lipid solution, for a TFR of 20 mL / min and FRR of 3 : 1. Suspended nanoparticles (4 mL) were collected and diluted by adding 46 mL PBS. To remove alcohol, the LNP suspension was transferred into a pre-hydrated Slide- A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed overnight against PBS. The particles were concentrated for 10 minutes at 2000 RCF in a Vivaspin™ ultrafiltration spin column with a 100K MWCO. The sample was collected from the upper filter and stored at -80° C in a 5% w / v sucrose solution.

[0113] Example E. Preparation of an LNP comprising MC3, cholesterol, DSPC, and ganglioside GM1 in a molar ratio of 46:37:13:4

[0114] Ganglioside GM1 (porcine) (ammonium salt), DLin-MC3-DMA, cholesterol, and DSPC were removed from storage at -20 °C and equilibrated to room temperature before the lipid materials were weighed out on an analytical balance. DLin-MC3-DMA, cholesterol, and DSPC were dissolved in appropriate volumes of ethyl alcohol to prepare solutions at concentrations of 50, 20, and 20 mg / mL, respectively. A 5 mg / mL solution of Ganglioside GM1 was prepared in methanol. Lipids solutions were warmed at 40 °C to ensure full solubilization before preparing the lipid packet. UltraPure™ DNase / RNase-Free distilled water was used to dissolve lyophilized Silencer™ GAPDH siRNA for a 1.0 mg / mL solution, as well as to prepare a 10 mM solution of citrate buffer (pH 3). A 0.026 mg / mL solution of RNA was made by adding 100 pL of siRNA solution to 3.736 mL of 10 mM citrate buffer.

[0115] Ganglioside GM1, DLin-MC3-DMA, cholesterol, and DSPC were combined at a molar ratio of 4:46:37: 13 for an N:P ratio of 6 and a total lipid concentration of 4 mM by adding 46.2, 22.7, 27.4, and 19.7 pL of the stocks, respectively, to 843 pL ethanol. The lipid and siRNA solutions were loaded into 1-mL and 5-mL BD Luer-Lock syringes and subjected to microfluidic mixing with the iNano L+ (Micro&Nano) using an S-SDM cartridge. Instrument settings were the following: 20 mL / min total flow rate, 1 :4 flow rate ratio, total volume 4.52 mL, and 0.5 mL start waste. The mixed nanoparticles were collected and then diluted to 40 mL in IX PBS to reduce concentration of organic solvents. The Nanoparticle solution was transferred to an Amicon Ultra centrifugal filter with 100 kDa molecular weight cut-off and spun at 2000 RCF to a volume of 1.2 mL. Aliquots for characterization were either stored at 4 °C or with 15% w / v sucrose at -80 °C.

[0116] The size, poly dispersity, and charge of the LNPs were measured with Malvern Panalytical’s Zetasizer Lab. The Z-average size was 102.5 nm with a poly dispersity index of 0.208; after freeze / thaw cycling the LNP size was 143.2 nm with 0.076 poly dispersity and - 29.76 mV Zeta potential. Encapsulation efficiency of the nucleic acid cargo was determined by conducting a Quant-iT™ Ribogreen assay and found to be 99.07% after overnight storage at 4 °C and 99.18% when stored at -80 °C. The ability to survive nebulization was confirmed using an Aeroneb® Lab nebulizer system with VMD 2.5 - 4.0 pm. Suspensions of LNPs in PBS were loaded into the nebulizer unit and aerosolized particles were collected. The postnebulized sizes of particles stored at 4 °C and particles freeze / thaw cycled at -80 °C were measured to be 103.9 nm and 145.2 nm with poly dispersity indices of 0.184 and 0.137, respectively. A Ribogreen assay demonstrated that the encapsulation efficiencies after nebulization were 93.85% and 97.05%.

[0117] Example F. Preparation of an LNP comprising MC3, Choi, DSPC, and GD3 in a molar ratio of 46:37.5:13:3.5

[0118] Ganglioside GD3, DLin-MC3-DMA, cholesterol, and DSPC were removed from storage at -20 °C and equilibrated to room temperature before the lipid materials were weighed out on an analytical balance. DLin-MC3-DMA, cholesterol, and DSPC were dissolved in appropriate volumes of ethyl alcohol to prepare solutions at concentrations of 50, 20, and 20 mg / mL, respectively. A 5 mg / mL solution of Ganglioside GD3 was prepared in methanol. Lipids solutions were warmed at 40 °C to ensure full solubilization before preparing the lipid packet. UltraPure™ DNase / RNase-Free distilled water was used to dissolve lyophilized AR-targeting sgRNA for a 3.25 mg / mL solution, as well as to prepare a 10 mM solution of citrate buffer (pH 3). A 0.026 mg / mL solution of total RNA was made by adding 15.4 pL of sgRNA solution and 50 pL of 1 mg / mL Cas9 mRNA to 3.77 mL of 10 mM citrate buffer. Ganglioside GD3, DLin-MC3-DMA, cholesterol, and DSPC were combined at a molar ratio of 3.5:46:37.5:13 for an N:P ratio of 6 and a total lipid concentration of 4 mM by adding 40.4, 22.7, 27.8, and 19.7 pL of the stocks, respectively, to 848 pL ethanol. The lipid and sgRNA / Cas9 mRNA solutions were loaded into 1 mL and 5 mL BD Luer-Lok syringes and subjected to microfluidic mixing with the iNano L+ (Micro&Nano) using an S-SDM cartridge. Instrument settings were the following: 20 mL / min total flow rate, 1 :4 flow rate ratio, total volume 4.6 mL, and 0.5 mL start waste. The mixed nanoparticles were collected and then diluted to 30 mL in IX PBS to reduce concentration of organic solvents. The Nanoparticle solution was transferred to an Amicon Ultra centrifugal filter with 100 kDa molecular weight cut-off and centrifuged at 2000 RCF for 15 minutes to a volume of 0.5 mL. Aliquots were stored with 15% w / v sucrose at -80 °C.

[0119] The size, poly dispersity, and charge of the LNPs were measured with Malvern Panalytical’s Zetasizer Lab. After one freeze-thaw cycle, the Z-average size was 132.9 nm with a poly dispersity index of 0.1396. Encapsulation efficiency of the nucleic acid cargo was determined by conducting a Quant-iT™ Ribogreen assay and found to be 98.53%. The ability to survive nebulization was confirmed using an Aeroneb® Lab nebulizer system with VMD 2.5 - 4.0 pm. Suspensions of LNPs in PBS were loaded into the nebulizer unit and aerosolized particles were collected. The post-nebulized particle size was measured to be 142.3 nm with polydispersity index of 0.074. A Ribogreen assay demonstrated that the encapsulation efficiency after nebulization was 97.35%.

[0120] Example G. Preparation of an LNP comprising MC3, Choi, DSPC, and GD3 in a molar ratio of 47.6:36.3: 12.4:3.6

[0121] A solution of the ionizable lipid MC3 at 50 mg / mL in ethanol was used. Stock solutions of cholesterol (Choi), and l,2-distearolyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A ganglioside GD3 stock solution at a concentration of 5 mg / mL was made in methanol. Lipid solutions were warmed at 40° C to ensure they were fully solubilized.

[0122] The lipid packet comprised MC3, Choi, DPSC, and GD3 in a molar ratio of 47.7:36.3: 12.4:3.6 and ethanol was added so the total concentration of lipids was 6 mM. For the cargo packet, a 0.039 mg / mL solution of F-Luc mRNA (Nl-my) was made by diluting F- Luc mRNA in 10 mM citrate buffer (pH 3). An N / P ratio of 6 / 1 was achieved by rapidly mixing the cargo and lipid solutions at a total flow rate of 20 mL / min and a flow rate ratio of 4: 1. The microfluidic mixing was performed with the iNano L+ instrument by Micro&Nano in an S-SDM cartridge. LNPs were collected, diluted 25X with PBS pH 7.4, and washed to remove alcohol. Nanoparticles were concentrated via centrifugation at 2000 RCF in an Amicon Ultra- 15 filter unit, then sterile filtered with a 0.22 pm syringe filter. Sterile sucrose was added to the LNPs at 15% w / v before storage at -80° C. An aliquot was later allowed to thaw at ambient temperature and taken for characterization. The size, polydispersity, and charge of the LNPs were measured with Malvern Panalytical’s Zetasizer Lab. The Z-average size was 105.6 nm with a poly dispersity index of 0.19, and the Zeta potential was -20.2 mV. Encapsulation efficiency of the nucleic acid cargo was determined by conducting a Quant- iT™ Ribogreen assay and found to be 96.8%. The LNP’s ability to survive nebulization was confirmed using an Aeroneb® Lab nebulizer system with VMD 2.5 - 4.0 pm. A suspension of LNPs in PBS was loaded into the nebulizer unit and aerosolized particles were collected. The particle size after nebulization was measured to be 123.4 nm with a polydispersity index of 0.245. A Ribogreen assay demonstrated that the encapsulation efficiency after nebulization was 93.2%.

[0123] Example H. Preparation of an LNP comprising ALC-0315, Choi, DSPC, and GD3 in a molar ratio of 47:35:12:6

[0124] A solution of the ionizable lipid ALC-0315 at 50 mg / mL in ethanol was used. Stock solutions of cholesterol (Choi), and l,2-distearolyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A ganglioside GD3 stock solution at a concentration of 5 mg / mL was made in methanol. Lipid solutions were warmed at 40° C to ensure they were fully solubilized.

[0125] The lipid packet comprised ALC-0315, Choi, DSPC, and GD3 in a molar ratio of 47:35: 12:6. Half of the GD3 was included in the lipid packet, and the remaining half was added to the aqueous cargo packet. Ethanol was added to the lipid packet so that the total concentration of lipids was 4 mM. For the cargo packet, a 0.02 mg / mL solution of F-Luc mRNA (N l -m\| / ) was made by diluting F-Luc mRNA in 10 mM citrate buffer (pH 3). An N / P ratio of 8 / 1 was achieved by rapidly mixing the cargo and lipid solutions at a total flow rate of 20 mL / min and a flow rate ratio of 4: 1. The microfluidic mixing was performed with the iNano L+ instrument by Micro&Nano in an S-SDM cartridge. LNPs were collected, diluted with PBS pH 7.4, and washed to remove alcohol. Nanoparticles were concentrated via centrifugation at 2000 RCF in an Amicon Ultra-15 filter unit, then sterile filtered with a 0.22 gm syringe filter. Sterile sucrose was added to the LNPs at 15% w / v before storage at -80° C. An aliquot of was later allowed to thaw at ambient temperature and taken for characterization. Five batches of this LNP formulation were prepared and pooled together for in vivo administration. The Z-average sizes after one freeze-thaw cycle were 135.7, 136.3, 150.9, 140.3, and 129.4 nm with polydispersity indices of 0.13, 0.05, 0.2, 0.11, and 0.11. The encapsulated RNA-weighted average size, zeta potential, and encapsulation efficiency were 136.5 nm, -21.4 mV, and 68.5%, respectively. The LNP’s ability to survive nebulization was confirmed using an Aeroneb® Lab nebulizer system. For instance, a suspension of the 5th batch in PBS was loaded into the nebulizer unit and aerosolized particles were collected. The particle size was measured to be 155.6 nm with a poly dispersity index of 0.209 after nebulization.

[0126] Example of a traditional LNP: Preparation of an LNP comprising MC3, Choi, DSPC, and PEG-lipid in a molar ratio of 50:38.5:10:1.5 to be used as a baseline for experimental comparison

[0127] Stock solutions of MC3, Choi, and DSPC (20 mg / mL) and a 10 mg / mL stock solution of DMG-PEG 2000 (PEG-lipid) were prepared by dissolving the appropriate amount of weighed lipids in ethyl alcohol. The lipid packet was made by combining 13.1 pL MC3 solution, 6.1 pL Choi solution, 3.2 pL DSPC solution, and 3.1 pL PEG-lipid solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM solution of citrate buffer (pH 3) was made by diluting the 0.5 M stock 50x with distilled water. To make the cargo packet (in excess) for a 6: 1 N:P ratio, 20 pL of the DasherGFP® mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0128] The lipid and cargo packets were loaded into 5 mL Luer-lock which were then fastened to a T-junction mixer fabricated in-house and secured onto two syringe pumps. T- mixer settings, controlled by KDS Legato software, were as follows: TFR = 20 mL / min; FRR = 3: 1; aqueous flow rate = 15 mL / min, lipid flow rate = 5 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was diluted by adding 36 mL PBS, then concentrated at 2000 RCF in a Vivaspin™ ultrafiltration spin column with a 100K MWCO. The sample was collected from the upper filter and stored at - 20° C in a 2.5% w / v sucrose solution. Nebulization and characterization of exemplary lipid nanoparticles

[0129] Nanoparticles suspensions were prepared in PBS such that they were between 20 to 200 pg / mL. An Aeroneb® Lab control module was used with an Aeroneb® Lab nebulizer unit that had the following specifications: flow rate > 0.1 mL / min; VMD between 2.5 pm - 4.0 pm; residual volume < 0.2 mL. The LNPs were loaded into the nebulizer unit and collected in a 50 mL conical tube.

[0130] The Z-average sizes and polydispersity indices (PDI) of the LNPs were measured with Malvern Panalytical’s Zetasizer Lab prior to and after nebulization. Encapsulation efficiencies of the nucleic acid cargo were determined by conducting Quant-iT™ Ribogreen assays. Total RNA concentrations were measured from LNP samples dissolved with 5% Triton-X, whereas free (non-encapsulated) RNA concentrations were measured using intact

[0131] LNPs suspended in IX TE buffer.

[0132] In vivo transfection of the lung in a murine model with aerosolized administration of the novel nebulized lipid nanoparticle platform:

[0133] Example G and Example H LNPs were administered via a mask nebulizer delivery system to male CD-I mice that were 6-8 weeks old. The animals were acclimated to the test facility for 72 h and health check observations were performed at least once daily. Clinical observations were performed prior to LNP administration and mice were closely monitored between dosing and the study terminal procedures. The LNPs were thawed at ambient temperature, and suspensions in saline were prepared so that the encapsulated RNA concentration was 20 pg / mL for Example G and 33.3 pg / mL for Example H. Dose volumes of 5 mL / animal for LNP G (n=2) and 3 mL / animal for LNP H (n=2) were used, such that the dose of encapsulated F-Luc mRNA delivered per animal was 100 pg.

[0134] Briefly, the delivery system from Kent Scientific comprised an Aeroneb® lab control module, a small VMD nebulizer unit, a nebulizer adapter, a small (7 mm) mask, and tubing. Mice were anesthetized with isoflurane prior to dosing and remained under anesthesia during inhaled LNP delivery. Adjustments were made to tubing connections to minimize condensation of the aerosolized particles prior to inhalation and the mouse head was aligned with the face mask to reduce loss of LNPs to the atmosphere. LNP solution that condensed in the adapter before delivery through the mask was collected with a pipet to be nebulized again. Since nebulized administration took 30-45 minutes, the mouse was placed on a heating pad. After dosing, the mice were returned to their cage housing for 4 h.

[0135] At 4 hours post-completion of dosing, mice were dosed subcutaneously with 0.2 mL of 15 mg / mL D-Luciferin. After 5-15 minutes, the animals were anesthetized with isoflurane and whole-body imaging of bioluminescence was performed with IVIS (in vivo imaging system). After in-life images were acquired, animals were euthanized by isoflurane overdose, then underwent exsanguination and whole-body intracardiac perfusion with 10 mL saline. The lungs, liver, and spleen were collected and subjected to ex-vivo IVIS imaging. The organs were fixed in 4% paraformaldehyde for 24 hours and then transferred to 70% ethanol for ambient storage.

[0136] In-life whole body and ex vivo bioluminescence were conducted on the tissues collected from the mouse having inhaled LNP-1 (Example G) and LNP-2 (Example H), respectively. The average radiance of luminescence for each tissue is listed in the table below:

[0137] Immunofluorescence histology:

[0138] The lung tissues were sectioned and stained for Luciferase, DAPI (nuclei), acetylated tubulin (ciliated cell marker), CD326 (epithelial cell marker), and CD31 (endothelial cell marker). Histology analysis demonstrated Cytofinity LNPs penetrated and transfected deep into lung tissues and that luciferase expressed colocalized with epithelial, endothelial, and ciliated cells. Figures 2A, 2B, and 2C showed the number of transfected endothelial, epithelial, and ciliated cells per square micrometer.

[0139] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. All articles, publications and patents referenced herein are incorporated by reference in their entirety.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A pharmaceutically acceptable lipid nanoparticle aerosol composition comprising a SA-bearing entity, preferably a ganglioside, and one or more of an ionizable lipid or cationic lipid, cholesterol or cholesterol derivative, and a phospholipid and optionally a PEG-lipid, said lipid nanoparticle has an average particle size of 5- 1,000 nm, preferably 20-500 nm, or 30-200 nm.

2. The composition of claim 1, wherein the molar percentage of ganglioside out of the total lipid composition is between 1-50%, 2-20%, 3-15%, 5-15% or about 10%.

3. The composition of claim 1, comprising ganglioside and one or more of an ionizable lipid, cholesterol and a phospholipid.

4. The composition of claim 3, wherein the molar percentage of cholesterol out of total lipid in the composition is 1-50%, 10-45%, or, more preferably between 15 and 40% of the total lipid composition.

5. The composition of claim 3, wherein the phospholipid is DSPC.

6. The composition of claim 3, wherein the molar percentage of phospholipid out of total lipid in the composition is 1-50%, 2-20%, 3-15%, 5-15% or about 10% of the total lipid composition.

7. The composition of claim 3, wherein the molar percentage of ionizable lipid out of total lipid in the composition is 1-60%, 10-55%, or, more preferably between 20 and 50%.

8. The composition of claim 1, further comprising an anionic active ingredient.

9. The composition of claim 8, wherein the anionic active ingredient is a nucleic acid.

10. The composition of claim 9, wherein the nucleic acid is a DNA, mRNA, a siRNA, a microRNA, an oligonucleotide, an aptamer, or a gene-editing agent such as a CRISPR-Cas9 construct.

11. The composition of any preceding claim, further characterized by an average particle size (Z-average) between 90% to 125%, such 100%, 105%, 110%, 115%, 120%, or 125%, of its original Z-average particle size of the LNP after the nebulization.

12. The composition of any preceding claim, further characterized by an encapsulation efficiency (EE) between 75% and 100%, such as 100%, 95%, 90%, 85% or 80%, of its original EE after nebulization.

13. A method for administration of a nucleic acid to a subject in need thereof comprising administering by inhalation to said subject the composition of any one of claims 1 to 12.

14. A method for the treatment of a disease or disorder in a subject in need thereof comprising administering by inhalation to said subject the composition of any one of claims 1 to 12.

15. The method of claim 14, wherein the disease is a lung disease.

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