Novel lipid nanoparticle compositions and uses thereof

Abstract

The present invention provides a lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)-containing lipid. The LNP formulation can effectively bind to cell surface Siglec, transfect specific cells in vitro, and target specific cells in vivo. The present invention also provides a method for using the LNP composition described herein for pharmaceutical applications. For example, the LNP provided herein is useful for intracellular delivery of nucleic acid therapeutic agents to a subject.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 356,557, filed June 29, 2022, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Nucleic acids, such as DNA, mRNA, siRNA, and antisense oligonucleotides (ASOs), have emerged as a new category of promising therapeutic agents for preventing and treating various diseases. However, there are still many unmet needs in converting nucleic acids into pharmaceuticals. For example, delivery of nucleic acid cargo to target cells relies heavily on viral vectors, which raises many safety and efficacy issues. Furthermore, safe, effective, and stable in vivo delivery of nucleic acid therapeutic agents remains a challenge. It is essential to incorporate nucleic acids into formulations that can protect nucleic acids from degradation and enable cellular uptake and intracellular cargo release.

[0003] Lipid nanoparticles (LNPs) are promising vehicles for delivering nucleic acid therapeutics because they can effectively protect and transport nucleic acid cargo into cells. For example, the LNP-based siRNA drug Onpattro has been approved by the US FDA for the treatment of polyneuropathy induced by hereditary transthyretin amyloidosis. An LNP-mRNA vaccine is currently in clinical use against COVID-19. These successes pave the way for the clinical development of many other nucleic acid-based therapies enabled by LNP delivery.

[0004] LNP formulations typically consist of nucleic acid cargo and four major lipid components: cationic or ionizable lipids, neutral phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. The cationic or ionizable lipids interact with the anionic nucleic acid during particle formation, facilitating cellular entry upon administration. The phospholipids stabilize the particle structure and, to some extent, regulate the biodistribution of LNPs. Cholesterol regulates cell membrane integrity and rigidity, thereby further stabilizing the particle and increasing the efficiency of in vivo delivery.

[0005] PEG lipids are also important components of LNP systems. PEG units play an important role in reducing particle size and minimizing particle aggregation. For systemically administered LNPs, PEG lipids reduce clearance mediated by the kidney and mononuclear phagocyte system (MPS), thereby extending blood circulation time, so that LNPs have sufficient time to reach their target site. In some cases, extended circulation can result in improved disease targeting mediated by the effect of enhanced permeability and retention (EPR).

[0006] Although conventional lipid nanoparticles have shown great success in delivering siRNA and mRNA, they have several drawbacks. For example, conventional LNPs have low efficiency in transfecting lymphocytes, such as T cells and natural killer (NK) cells, as well as macrophages. T cells, NK cells, and macrophages are the targeted cells for many therapeutic modalities, such as CAR-T, CAR-NK, and CAR-macrophage therapy. Conventional LNPs also suffer from deficiencies in in vivo targeting, particularly targeting immune cells and tumor cells. To treat inflammatory diseases, it is also important to target immune cells and the complement system, which are the source of inflammation. However, this cannot always be achieved using conventional LNPs.

[0007] In addition, systemically administered LNPs tend to accumulate primarily in the liver. Consequently, most LNP-based therapies target the liver and liver-related diseases. Therefore, "extrahepatic" delivery for the treatment of non-liver-related diseases is a major unmet need in the biopharmaceutical field.

[0008] Recently, it has been reported that PEG-lipids in conventional LNP formulations can lead to the formation of anti-PEG antibodies, which can cause anaphylaxis, accelerated blood clearance (ABC), and undesirable immunogenicity. Therefore, it is desirable to replace PEG-lipids in LNP formulations with non-immunogenic entities.

[0009] These drawbacks limit the application of LNPs in certain areas, such as adoptive cell therapy, in vivo cell engineering, and gene therapy.

[0010] Sialic acid, also known as N-acetylneuraminic acid, is a nine-carbon sugar that binds to sialic acid-binding immunoglobulin-like lectins (Siglecs). Sialic acid has three main derivatives: N-acetylneuraminic acid (Neu5Ac), N-acetylneuraminic acid hydroxyalkyl (Neu5Gc), and 3-deoxy-D-glycero-D-galacto-nonylketose (Kdn). Other sialic acid derivatives are further derived from these major derivatives. One important sialic acid derivative is ganglioside, which is found in the brain and other human tissues.

[0011] Siglecs, mostly expressed by various immune cells, primarily contain intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which can mediate inhibitory signals upon binding to sialic acid and activate downstream inhibitory signaling by recruiting the tyrosine phosphatases SHP-1 and SHP-2. Some Siglecs contain intracellular immunoreceptor tyrosine-based activation motifs (ITAMs), which can mediate stimulatory signals upon binding to sialic acid and activate downstream stimulatory signaling. Sialic acid can also regulate alternative pathways of complement activation. The major serum protein complement factor H recognizes sialic acid as a "self" marker and helps inhibit C1q / C3b fragment activation. Sialic acid also binds to carbohydrate-binding lectins, which are overexpressed in several types of cancer.

[0012] Abnormal interactions between sialic acid and Siglecs are associated with several pathologies, including infection, autoimmunity, inflammation, and cancer. Therefore, binding to Siglecs on certain cell types with chemical or biological entities containing sialic acid residues may be therapeutically beneficial for modulating immune inhibition or activation for the treatment of such conditions. Binding to cell surface Siglecs also enables endocytosis, thereby enhancing cell targeting and intracellular delivery of therapeutic agents.

[0013] However, it is difficult to deliver such molecular entities of sialic acid to target cells in vivo. A common strategy is to attach sialic acid or polysialic acid molecules onto the surface of nanoparticles, so that the nanoparticles can carry the sialic acid entities to target cells.

[0014] Furthermore, when sialic acid binds to Siglec on certain types of immune cells, a chemical entity containing a sialic acid moiety can be attached to the particle as a ligand to guide the particle to the immune cell and bind to Siglec on the cell. Such binding can facilitate the particle to enter the cell via receptor-mediated endocytosis. In this manner, nanoparticles loaded with a therapeutic agent and coated on the surface with sialic acid or a sialic acid-containing entity can target immune cells and deliver the therapeutic agent to the cell.

[0015] The conventional method for attaching sialic acid to the surface of lipid nanoparticles is by chemical conjugation.For example, sialic acid molecules can be functionalized with a reactive group that can form a covalent bond with another reactive group on the surface of nanoparticles.However, this method suffers from low conjugation efficiency and side reactions that produce undesirable by-products in pharmaceutical formulations.

[0016] Therefore, there is an unmet need for novel methods to attach sialic acid-containing ligands to the surface of nanoparticles, especially LNPs, to facilitate cell transfection and targeting via binding of Siglecs by the SA-containing ligands on LNPs. Summary of the Invention

[0017] Summary of the Invention The present invention in part provides lipid nanoparticle (LNP) formulations comprising at least one sialic acid (SA)-containing lipid, and the LNP formulations can effectively bind to cell surface Siglec, transfect specific cells in vitro, and target specific cells in vivo.The present invention also provides the method of using the LNP compositions described herein for pharmaceutical applications.For example, the LNPs provided herein are useful for intracellular delivery of nucleic acid therapeutic agents to subjects.

[0018] The SA-containing lipids may be of synthetic or natural origin and may contain multiple sialic acid residues selected from the group consisting of Neu5Ac, Neu5Gc, and Kdn, or combinations thereof. The SA-containing lipids may have molecular weights of 200-500,000, 500-100,000, and 1,000-50,000 Da.

[0019] In some embodiments, the SA-containing lipid is formed by coupling a chemical entity comprising at least one SA unit with a fatty acid, where the fatty acid can be a saturated or unsaturated fatty acid and can have 2 to 100, preferably 10 to 30, and more preferably 13 to 21 carbons. In some embodiments, the SA-containing lipid can be linear and can contain only one lipid unit.

[0020] In some embodiments, the SA-containing lipid may be branched and may contain multiple lipid units.

[0021] In some embodiments, the SA-containing lipid is a naturally occurring lipid.

[0022] In some embodiments, the SA-containing lipid is a ganglioside, a ganglioside derivative, a mimetic, or a combination thereof.

[0023] Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked to the sugar chain. Gangliosides can be of synthetic or natural origin.

[0024] The gangliosides used in the LNP compositions of the invention can be gangliosides containing one SA unit, e.g., GM1, GM2, GM3, asialo-GM1, GA1, asialo-GM2, GA2, or gangliosides containing two SA units, e.g., GD1a, GD1b, GD2 and GD3, or gangliosides containing three SA units, e.g., GT1a, GT1b, GT1c, OAc-GT1b, GT3, or gangliosides containing four SA units, e.g., GQ1.

[0025] Other non-limiting 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 from Avanti Polar Lipids, Birmingham, AL.

[0026] For gangliosides containing only a single SA unit (e.g., in the case of GM1, GM2, and GM3), SA can be linked to its adjacent sugar ring via an α2,3, α2,6, α2,8, or α2,9 bond. For gangliosides containing only one SA unit, an α2,3 bond is preferred in the present invention.

[0027] The present invention also provides LNP formulations comprising at least one SA-containing lipid (e.g., ganglioside) as described herein, and methods of using the same. In addition to nucleic acid cargo (e.g., DNA, mRNA, or siRNA), the LNP formulations may also contain other types of lipids, including, but not limited to, cationic or ionizable lipids, cholesterol, PEG-lipids, and phospholipids.

[0028] Cationic lipids are typically pharmaceutically acceptable and can be natural or synthetic lipids. Examples of cationic lipids include ammonium lipids or lipids characterized by a positively charged nitrogen moiety. For example, cationic lipids can be substituted with a tertiary ammonium group, such as trialkylammonium, preferably trimethylammonium. Cationic lipids can be further substituted with one or more substituted or unsubstituted long-chain alkyl or alkenyl groups, such as G4 to C20 alkyl or alkenyl groups. Examples of commonly used lipids include polyvalent cationic lipids, DOTMA, ethyl PC, DDAB, pH-sensitive lipids, dioleoyl-3-trimethylammonium propane (DOTAP), DC cholesterol, and GL67.

[0029] Ionizable lipid is a class of lipid molecules that are neutral and non-ionic at physiological pH, but are protonated and positively charged at lower pH.Ionizable lipid can also form complexes with SA-containing entities, promoting endosomal escape and reducing toxicity.Commercially available examples of ionizable lipid include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, ALC-0315, SM-102, DODMA and DODAP.

[0030] Other chemical entities commonly used in lipid nanoparticle (LNP) formulations, such as structural lipids, PEGylated lipids, cholesterol, phospholipids, etc., can be added to the nanoparticle formulations of the present invention to enhance the stability, functionality, and other performance properties of complex nanoparticles.

[0031] The present invention provides lipid nanoparticle compositions comprising at least gangliosides capable of binding to cell surface Siglecs for the treatment of diseases or disorders, such as cancer, autoimmune and inflammatory diseases. The present invention also includes a step of administering the lipid nanoparticle composition to a subject.

[0032] The present invention further provides a method for delivering an active pharmaceutical ingredient (e.g., a nucleic acid) to a cell in a subject in need thereof, comprising administering to the subject the composition.

[0033] The present invention further relates to a method for treating a disease or disorder, such as cancer or an autoimmune or inflammatory disease, in a subject in need of such treatment, comprising administering to said subject a composition of the present invention.

[0034] The present invention also relates to methods for the preparation of lipid nanoparticles that are capable of delivering nucleic acid cargo to specific cells in vitro and targeting specific cells in vivo upon systemic administration.

[0035] The present invention also provides lipid nanoparticle compositions that are free of PEG lipids to reduce or eliminate the potential for anti-PEG formation, allergic reactions, accelerated blood clearance, and undesirable immunogenicity. [Brief explanation of the drawings]

[0036] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] FIG. 1A is a cartoon of an LNP nanoparticle of the present invention. [Figure 1B] FIG. 1B is a schematic diagram of an LNP nanoparticle and its interaction with a target cell. [Figure 2] 2A and 2B are graphs of GAPDH knockdown in cells using siRNA-loaded Sia-bearing lipid LNPs. DETAILED DESCRIPTION OF THE INVENTION

[0037] Detailed Description of the Invention A description of preferred embodiments of the present invention follows.

[0038] definition As used herein, "pharmaceutically acceptable" includes those compounds, materials, compositions and / or dosage forms that are within the scope of sound medical judgment and that, when contacted with the tissues of humans and animals at the concentrations, dosages or amounts present in the product, do not produce undue toxicity, irritation, allergic response or other problem or complication, are suitable for medical or veterinary use, commensurate with a reasonable benefit / risk ratio. Preferably, pharmaceutically acceptable materials (e.g., polymers, excipients, surfactants, solvents or microparticles / nanoparticles made therefrom) are suitable or approved for human medical use.

[0039] As used herein, a "nanoparticle" is preferably roughly round, spherical, or spherical-like in shape and generally falls within a size range of, for example, about 1-1,000 nm, about 10-1,000 nm, or about 50-1,000 nm, or about 100-500 nm, as measured, for example, by dynamic light scattering. The subject nanoparticles can also include particles that do not tend to aggregate into clumps in vivo.

[0040] Particle size and size distribution can be measured by dynamic light scattering instruments, such as a Malvern Zetasizer. Particle size is typically reported as Z-average diameter. Alternative techniques include, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, light diffraction, and disk centrifugation. The term "nanoparticle" is not intended to imply any particular shape restriction. Such particles include, but are not limited to, those generally having a polygonal or spherical geometry. Preferred particles are characterized by a spherical geometry, typically produced by an emulsion-based encapsulation process. It is understood that the terms "microparticle" and "nanoparticle" are used interchangeably herein unless a specific description of size is attached. For example, the term "microparticle" is intended to encompass "nanoparticles," as well as when described as "microparticles and / or nanoparticles," unless the context requires otherwise.

[0041] Although individual nanoparticles need not be of uniform size, they are generally of a size sufficient to induce endocytosis in antigen-presenting cells (APCs), other MPS cells, or lymphocytes. Preferably, the subject nanoparticles have a diameter sufficient to induce phagocytosis in antigen-presenting cells (APCs), macrophages, NK cells, T cells, and / or other MPS cells.

[0042] The term "particle" encompasses both nanoparticles and microparticles.

[0043] As used herein, "a" or "an" means one or more, unless specified otherwise.

[0044] As used herein, "about" generally means up to plus or minus 10% of the particular term it modifies.

[0045] The terms "sialic acid residue" and "sialic acid moiety" and their plural referents, etc. are used interchangeably herein.

[0046] As used herein, the term "subject" is used to mean an animal, preferably a mammal, such as a human or non-human. The terms "patient" and "subject" may be used interchangeably herein.

[0047] "Treatment" or "therapy" of a subject refers to any type of intervention or process performed on a subject or administration of an active agent to a subject for the purpose of reversing, alleviating, ameliorating, suppressing, slowing down, or preventing the onset, progression, onset, severity, or recurrence of symptoms, complications, conditions, or biochemical markers associated with a disease. As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") includes clinical intervention to alter the natural course of disease in the individual being treated and can be performed either prophylactically or during the course of clinical illness. Desired effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, improvement or mitigation of disease state, and remission or improved prognosis. In some embodiments, the combinations of the present invention are used to delay the onset of disease or slow the progression of disease.

[0048] Gangliosides Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked on a sugar chain. They form lipid platforms in the outer leaflet of cell membranes, especially in neuronal cells of the central nervous system. Gangliosides have been found to be very important molecules in immunity where they participate in cell proliferation, differentiation, adhesion, signal transduction, cell-cell interactions, tumorigenesis, and metastasis. More than 60 gangliosides are known. [ka] [ka]

[0049] Gangliosides can be named based on the number of SA units they contain in the molecule. Thus, gangliosides with one SA unit are named "GM", such as GM1, GM2, and GM3. Here, "G" stands for "ganglioside" and "M" stands for "mono". Similarly, "GD", "GT", and "GQ" refer to gangliosides with two ("di"), three ("tri"), and four ("quadruple") units, respectively. For illustrative purposes, the structures of gangliosides GM1 and GD3 are shown above.

[0050] The SA units in ganglioside molecules can bind to Siglecs expressed on various types of cells, such as immune cells and tumor cells. Therefore, lipid nanoparticles incorporating ganglioside molecules on their surfaces can facilitate the binding of Siglecs and the delivery of nucleic acid cargoes into cells via receptor-mediated endocytosis for disease treatment.

[0051] In a specific embodiment of the present invention, gangliosides having SA units and α2,8 linkages can bind to Siglec-7 expressed on natural killer (NK) cells and can be incorporated into certain lipid nanoparticle formulations to facilitate delivery of nucleic acid cargo to NK cells. Non-limiting examples of such gangliosides include GM2b, GD3, GT1a, GT3, and GQ1b.

[0052] In another embodiment, lipid nanoparticles incorporating other gangliosides can be used to target other immune cells, such as monocytes, macrophages, dendritic cells, B cells, neutrophils, T cells, etc.

[0053] Also, because sialic acid binds to several receptors on tumor cells, ganglioside-containing LNPs can be enhanced to target tumor sites via the high-avidity binding of sialic acid to lectins.

[0054] Gangliosides may be added to the formulation in an amount of at least about 0.1% (molar percentage total lipid in the composition) (including any PEG-lipids that may be added as described in more detail below), preferably 0.1-50%, 0.5-20%, or 1-10%. Gangliosides preferably have 1, 2, 3, 4, or more sialic acid units.

[0055] Cationic and Ionizable Lipids Thus, lipids can have a positive or partial positive charge at physiological pH. Such lipids can be referred to as cationic or ionizable (amino) lipids. Lipids can also be zwitterionic, i.e., neutral molecules with both positive and negative charges.

[0056] In some embodiments, the lipid is selected from the group consisting of dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (diethanami-ne) (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL26), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL27), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL28), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL29), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL30), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL31), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL32), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL33), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL34), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL35), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL36), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL37), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (KL DLin-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-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]-dioxolane (DLin-KC2-DMA), 1,2-Dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-

[0039] The compound may be selected from (2R)-2-({8-[(3.beta.)-cholest-5-en-3-yloxy]octyl})oxy)-N,N-dimethyl-3-[(9-Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA (2R)), (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-1-amine (octyl-CLinDMA (2S)).

[0057] Ionizable lipids are a class of lipid molecules that are neutral and non-ionic at physiological pH, but are protonated and positively charged at lower pH.Ionizable lipids can also form complexes with SA-containing entities, promoting endosomal escape and reducing toxicity.Commercially available examples of ionizable lipids include DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, DODMA, DODAP, ALC-0315, SM-102, SS-OP, SS-EC, etc.

[0058] The cationic lipid may be added to the formulation in an amount of at least about 10% (molar percentage total lipid in the composition) (including any PEG lipids that may be added as described in more detail below), preferably at least 20%, at least 30%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, or at least 49%. For example, the cationic lipid may be present in an amount of 25-75% (molar percentage total lipid), preferably 30-60%, and more preferably 45-55%.

[0059] PEG lipids 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 (such as 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, PEG lipids can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC or PEG-DSPE lipids.

[0060] The PEG lipids may be added to the formulation in an amount of at least about 1% (molar percentage total lipid in the composition) (including any PEG lipids that may be added as described in more detail below), at least about 0.1% (molar percentage total lipid in the composition) (including any PEG lipids that may be added as described in more detail below), preferably 0.1-50%, 0.5-20%, or 1-10%.

[0061] It should be noted that in the present invention, the addition of PEG-lipid is optional.Because PEG-lipid may cause the formation of anti-PEG, which may cause anaphylaxis, accelerated blood clearance and undesired immunogenicity, it is beneficial to construct LNP without PEG-lipid.The ganglioside in the LNP of the present invention can stabilize nanoparticles and prolong the circulation time of LNP in blood circulation, allowing nanoparticles to find their targeting site.Without being bound by any theory, it is believed that ganglioside exists on the surface of LNP to create a hydrophilic particle surface, thereby avoiding uptake by MPS and prolonging circulation.

[0062] The hydrophilic nature of the LNPs of the present invention can also reduce the formation of protein coronas, such as those formed by apolipoproteins, such as Apo-E. The Apo-E corona is believed to direct nanoparticles to the liver. Therefore, the LNP compositions of the present invention can also be used to deliver therapeutic agents to tissues and organs other than the liver. Such "extrahepatic" organs include, but are not limited to, the spleen, kidneys, lungs, heart, bone marrow, lymph nodes, brain, skin, ears, and eyes.

[0063] Other chemical entities commonly used in lipid nanoparticle (LNP) formulations, such as structural lipids, cholesterol, phospholipids, etc., can be added to the nanoparticle formulations of the present invention to increase the stability, functionality, and other performance characteristics of the LNP formulations.

[0064] For example, cholesterol may be present in an amount of 5-60% (molar percentage total lipids), preferably 10-55%, more preferably 25-50%.

[0065] The phospholipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-m al), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In a preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC). The phospholipid may be present in an amount of 1-50% (molar percentage total lipid), preferably 2-40%, and more preferably 5-15%.

[0066] activator The described LNPs can further comprise an active agent. The composition can include an API. The API can be encapsulated within the LNP. The amount of API can be about 0.01 to about 50% (w / w) of the nanoparticle, 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. The lipid to nucleic acid ratio can be about 1 to 20, preferably 3 to 15, and more preferably 5 to 10.

[0067] In some aspects, the active agent is advantageously a drug (also referred to herein as an active pharmaceutical ingredient, or API). However, non-therapeutic active agents can also be included as part of the particle according to the method. For example, agents useful in diagnostics, agriculture, cosmetics, personal products, household products, industrial chemicals, dyes, fluorescers, or colorants, etc., can be included. Preferred active agents are oligonucleotides, nucleic acid molecules and mimetics thereof, such as DNA, RNA, PNA, siRNA, microRNA, oRNA, antisense, oligonucleotides, aptamers, and combinations thereof. It is understood that API can be replaced with non-therapeutic compounds, such as diagnostic, agricultural, or chemical agents. Therefore, in each instance where the term API is used, it will be understood that the term "active agent," including diagnostic, agricultural, or chemical agents, can be used instead. The terms "API" and "cargo" are used interchangeably herein.

[0068] Particle preparation LNP can be produced by co-precipitation of lipids and nucleic acids. Generally, SA-containing lipids (e.g., gangliosides) can be dissolved together with other lipids in a water-miscible organic solvent, such as alcohol. Any nucleic acid API can be dissolved in an aqueous medium or an organic solvent. The two solutions are then combined. Preferably, the aqueous solution and the organic solution are combined slowly (e.g., dropwise) or by mixing (e.g., via a microfluidic device) to cause nanoprecipitation. For example, a small amount of organic solution can be added to the aqueous phase while mixing.

[0069] Particles can also be produced using pre-assembled devices such as a T-mixer or automated microfluidic devices such as Precision Nanosystems' NanoAssemblr and Particle Works' Automated Nanoparticle System.

[0070] Exemplary solvents that are miscible with water include methanol, ethanol, acetone, tetrahydrofuran (THF), acetonitrile, dimethylsulfoxide (DMSO), and dimethylformamide (DMF).

[0071] The solvent is then removed and / or the particles recovered, for example by evaporation, solvent exchange, centrifugation or filtration, dialysis, tangential flow filtration, followed by dehydration, for example concentration or lyophilization.

[0072] The aqueous solution may optionally contain surfactants, including organic or inorganic pharmaceutical excipients, various polymers, oligomers, natural products, nonionic, cationic, zwitterionic, or ionic surfactants, and mixtures thereof. The surfactants may include acidic buffers, such as citrate, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polysorbate (Tween series) surfactants, PEO-PPO-PEO (polyethylene oxide-polypropylene oxide-polyethylene oxide) triblock copolymers (Pluronic series or Poloxamer series) surfactants, or t-octylphenyl-polyethylene glycol (Triton X-100) surfactants, or salts, derivatives, copolymers, or mixtures thereof.

[0073] particle size The size of the subject nanoparticles is about 1 nm to about 10 μm, preferably about 10 nm to about 2 μm, more preferably about 20 nm to about 1 μm, and most preferably about 40 nm to about 500 nm. For example, the nanoparticles can have an average size of about 50 to 900 nm, e.g., about 50, 75, 100, 300, 500, 700, or 900 nm.

[0074] As used herein, particle size may be determined by any conventional particle size measurement technique known to those skilled in the art, including, for example, sedimentation field flow fractionation, photon correlation spectroscopy, light scattering, dynamic light scattering, light diffraction, and disk centrifugation.

[0075] Pharmaceutical Composition One aspect of the present invention provides pharmaceutical compositions comprising the subject lipid nanoparticles, and optionally comprising a pharmaceutically acceptable carrier or excipient.Preferably, these compositions optionally further comprise one or more additional therapeutic agents.Alternatively, the subject particles of the present invention can be administered to a patient in need thereof in combination with the administration of one or more other therapeutic agents.For example, the additional therapeutic agent for co-administration with the compound of the present invention or inclusion in a pharmaceutical composition can be an approved anti-inflammatory agent, immunotherapeutic agent, or chemotherapeutic agent, or any one of several drugs approved by the Food and Drug Administration.It is also understood that certain subject particles of the present invention can be present in free form for treatment, or, where appropriate, as its pharmaceutically acceptable derivative.

[0076] Preferably, the pharmaceutical compositions of the present invention further comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents or other liquid vehicles, dispersing or suspending aids, surface active agents, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like, as appropriate for the particular dosage form desired. Remington's Pharmaceutical Sciences, 16th Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional carrier medium is incompatible with the compounds of the present invention, such as by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0077] Some examples of materials that may serve as pharmaceutically acceptable carriers include, but are not limited to, sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil; safflower oil, sesame oil; olive oil; corn oil, and soybean oil; glycols; such as propylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffer solution; other non-toxic, compatible lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening agents, flavoring and perfuming agents, preservatives and antioxidants, adjuvants, such as, but not limited to, Alum, MF59, AS04 and CPG, can also be present in the composition, according to the judgment of the formulator.

[0078] 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, cow, bull, cow, sheep, goat, pig, camel, etc.), a rodent (rabbit, hamster, mouse, rat, etc.), or a pet (cat, dog).

[0079] In one embodiment, the method comprises administering a subject composition or pharmaceutical composition comprising a subject microparticle or nanoparticle (e.g., a carboxylated particle) by any suitable means or route, such as orally, nasally, intravenously, intramuscularly, ocularly, transdermally, or subcutaneously. In a particular embodiment, the particle is administered intranasally. In yet another embodiment, the particle is administered intravenously. [Example]

[0080] Example Example 1. Preparation of lipid nanoparticles containing poly(A) and ganglioside GM3 Ganglioside GM3 was dissolved in a solvent mixture consisting of 1 part ethanol and 3 parts methanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GM3 were mixed in a molar ratio of 50:38.5:10:1.5. Poly(A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into two syringes mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the poly(A) solution, achieving an N:P ratio of 5.79. The resulting lipid nanoparticles were found to have a Z-average particle size of 93.7 nm and a zeta potential of -11.9 mV.

[0081] Example 2. Preparation of lipid nanoparticles containing poly(A) and ganglioside GM3 Ganglioside GM3 was dissolved in a solvent mixture consisting of 1 part ethanol and 3 parts methanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GM3 were mixed in a molar ratio of 48.5:37.3:9.7:4.5. Poly(A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into two syringes mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the poly(A) solution, achieving an N:P ratio of 5.79. The resulting lipid nanoparticles were found to have a Z-average particle size of 122.3 nm and a zeta potential of -14.6 mV.

[0082] Example 3. Preparation of lipid nanoparticles containing poly(A), PEG lipids, and ganglioside GM3 Ganglioside GM3 was dissolved in a solvent mixture consisting of 1 part ethanol and 3 parts methanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GM3 were mixed in a molar ratio of 49.2:37.9:9.8:1.5:1.5. Poly(A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) was dissolved in 10 mM citrate buffer (pH 3) at an N:P ratio of 5.79. The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into two syringes mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the poly(A) solution, achieving an N:P ratio of 5.79. The LNPs were then dialyzed against PBS for 12 hours. The resulting lipid nanoparticles were found to have a Z-average particle size of 65.7 nm and a zeta potential of -2.54 mV.

[0083] Example 4. Preparation of lipid nanoparticles containing poly(A), PEG lipids and ganglioside GD3 Ganglioside GD3 was dissolved in a solvent mixture consisting of 1 part water and 3 parts ethanol. Other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 49.2:37.9:9.8:1.5:1.5. Poly(A) (MW 700,000-3,500,000, chain length 2,100-10,000 nucleotides) was dissolved in 10 mM citrate buffer (pH 3). The two solutions were introduced into a T-mixer, and the resulting LNP was dialyzed against PBS for 12 hours.

[0084] Example 5. Preparation of lipid nanoparticles containing mRNA, PEG-lipids and ganglioside GD3 Ganglioside GD3 is dissolved in a solvent mixture consisting of 1 part water and 3 parts ethanol. Other lipids are dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 are mixed in the appropriate molar ratio. GFP mRNA is dissolved in aqueous buffer. The two solutions are introduced into a T-mixer, and the resulting LNP is dialyzed against PBS for 12 hours.

[0085] Example 6. Preparation of lipid nanoparticles containing siRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 3 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000, and GD3 were mixed in a molar ratio of 49:37.5:9:1.5:3. Silencer TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-lok syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 130.3 nm and a zeta potential of -5.75 mV. The encapsulation efficiency was 90.8%, as measured by the Ribogreen assay.

[0086] Example 7. Preparation of lipid nanoparticles containing siRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 5 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000, and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Silencer TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 9.0 ml, and the volume of the lipid solution was 3.0 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 101.9 nm and a zeta potential of -32.56 mV. The encapsulation efficiency was 92.8%, as measured by the Ribogreen assay.

[0087] Example 8. Preparation of lipid nanoparticles containing siRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 10 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000, and GD3 were mixed in a molar ratio of 45:35:8.5:1.5:10. Silencer TMGAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 9.0 ml, and the volume of the lipid solution was 3.0 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 119.9 nm and a zeta potential of -35.68 mV.

[0088] Example 9. Preparation of lipid nanoparticles containing siRNA, ionizable lipid MC3, cholesterol, phospholipids, and 5 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 49:37:9:5. Silencer TM GAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 2.5x the volume of PBS solution, dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 184.9 nm and a zeta potential of -32.55 mV. The encapsulation efficiency was 95.0%, as measured by the Ribogreen assay.

[0089] Example 10. Preparation of lipid nanoparticles containing ionizable lipid MC3, cholesterol, phospholipids, and 5 mol% of ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 49:37:9:5. A blank 10 mM citrate buffer (pH 3) was prepared without dissolving any RNA. The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the buffer solution. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 116.8 nm.

[0090] Example 11. Preparation of lipid nanoparticles containing mRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 3 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 49:37.5:9:1.5:3. GFP-mRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 81.5 nm and a zeta potential of -33.46 mV. The encapsulation efficiency was 92.5% as measured by the Ribogreen assay.

[0091] Example 12. Preparation of lipid nanoparticles containing mRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 5 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 48:36.5:9:1.5:5. GFP-mRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 106.9 nm and a zeta potential of -33.08 mV. The encapsulation efficiency was 92.6% as measured by the Ribogreen assay.

[0092] Example 13. Preparation of lipid nanoparticles containing mRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 10 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 45:35:8.5:1.5:10. GFP-mRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 101.5 nm and a zeta potential of -35.22 mV. The encapsulation efficiency was 85.3% as measured by the Ribogreen assay.

[0093] Example 14. Preparation of lipid nanoparticles containing mRNA, ionizable lipid MC3, cholesterol, phospholipids, and 10 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 46:35:9:10. GFP-mRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into a 10 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 40 ml of PBS solution, stirred for 1 hour, and then dialyzed against PBS for 6 hours. The nanoparticle suspension was concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 164.0 nm and a zeta potential of -50.03 mV. The encapsulation efficiency was 86.0% as measured by the Ribogreen assay.

[0094] Example 15. Preparation of lipid nanoparticles containing siRNA, ionizable lipid SM-102, cholesterol, phospholipid, PEG lipid, and 5 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. SM-102, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Anti-GAPDH siRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 74.9 nm and a zeta potential of -25.12 mV. The encapsulation efficiency was 71.1% as measured by the Ribogreen assay.

[0095] Example 16. Preparation of lipid nanoparticles containing siRNA, ionizable lipid ALC-0315, cholesterol, phospholipid, PEG lipid, and 5 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. ALC-0315, cholesterol, DSPC, DMG-PEG2000, and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Anti-GAPDH siRNA was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 73.6 nm and a zeta potential of -30.88 mV. The encapsulation efficiency was 35.1% as measured by the Ribogreen assay.

[0096] Example 17. Preparation of lipid nanoparticles containing siRNA, ionizable lipid MC3, PEG lipid, and 5% ganglioside GD3 using Nanoassembly Ignite Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000, and GD3 were mixed in a molar ratio of 49:36.5:8:1.5:5. Silencer TMGAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 1.5 ml, and the volume of the lipid solution was 0.5 ml. The cargo solution was loaded into a 3 mL Luer-lock syringe, and the lipid solution was loaded into a 1 mL Luer-lock syringe mounted on a Nanoassembly Ingite (Precision Nanosystems, Vancouver, Canada). The flow rates were set at 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 90.3 nm and a zeta potential of -25.57 mV.

[0097] Example 18. Preparation of lipid nanoparticles containing fluorescently tagged GFP-mRNA, ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 3 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, PEG2000, and GD3 were mixed in a molar ratio of 49:37.5:9:1.5:3. Cy5-tagged GFP-mRNA (APExBIO's EZ Cap Cy5 EGFP mRNA) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into 10 mL Luer-Lock syringes mounted on syringe pumps and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 20 ml of PBS, then dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at −20° C. The resulting lipid nanoparticles were found to have a Z-average particle size of 101.0 nm, a PDI of 0.164, and a zeta potential of −23.7 mV. The encapsulation efficiency was 84.9%, as measured by the Ribogreen assay.

[0098] Example 19. Preparation of lipid nanoparticles containing fluorescently tagged GFP-mRNA, ionizable lipid MC3, cholesterol, phospholipids, and 10 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. MC3, cholesterol, DSPC, and GD3 were mixed in a molar ratio of 46:35:9:10. Cy5-tagged GFP-mRNA (APExBIO's EZ Cap Cy5 EGFP mRNA) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 7.5 ml, and the volume of the lipid solution was 2.5 ml. The two solutions were loaded into 10 mL Luer-Lock syringes mounted on syringe pumps and then introduced into a T-mixer at a flow rate of 8 ml / min for the lipid solution and 24 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 20 ml of PBS, then dialyzed against PBS for 6 hours, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at −20° C. The resulting lipid nanoparticles were found to have a Z-average particle size of 226.3 nm, a PDI of 0.251, and a zeta potential of −43.58 mV. The encapsulation efficiency was 95.8%, as measured by the Ribogreen assay.

[0099] Example 20. Preparation of lipid nanoparticles containing siRNA, ionizable SS-OP, cholesterol, phospholipids, and 10 mol% ganglioside GD3 Ganglioside GD3 was dissolved in methanol. All other lipids were dissolved in ethanol. SS-OP (obtained from NOF Corporation), cholesterol, DSPC, and GD3 were mixed in a molar ratio of 46:35:9:10. Silencer TMGAPDH siRNA (ThermoFisher) was dissolved in 10 mM citrate buffer (pH 3). The volume of the citrate buffer was 3.0 ml, and the volume of the lipid solution was 1.0 ml. The two solutions were loaded into a 5 mL Luer-Lock syringe mounted on a syringe pump and then introduced into a T-mixer at a flow rate of 5 ml / min for the lipid solution and 15 ml / min for the RNA solution, achieving an N:P ratio of 6:1. After mixing, the nanoparticle suspension was mixed with 30 ml of PBS, concentrated using an Amicon® ultracentrifugal filtration device at 2000 rcf, and prepared with 5% w / v sucrose for storage at -20°C. The resulting lipid nanoparticles were found to have a Z-average particle size of 253.6 nm, a PDI of 0.157, and a zeta potential of -43.59 mV.

[0100] Example 21: In vitro siRNA knockdown of GAPDH in primary human NK cells, macrophages, and CD8+ T cells using LNPs containing 3% GD3 Lipid nanoparticles containing Silencer™ GAPDH siRNA (ThermoFisher), ionizable lipid MC3, cholesterol, phospholipid, PEG lipid, and 3 mol% ganglioside GD3 were prepared as described in Example 6. The prepared lipid nanoparticles were incubated with primary human NK cells, CD8+ T cells, and macrophages at 30 nM and 90 nM siRNA concentrations. Samples were collected at 12 and 48 hours, and qPCR analysis of GAPDH gene expression was performed. The data were normalized to ActinB gene expression. Detailed experimental conditions are described as follows:

[0101] A. Cell culture a. Primary human CD8+ T cells PBC-1041, NK cells PB56C-2, and M1 macrophages PBM1C-MON-3 b. OptiMEM I Reduced Serum Medium with Pen-Strep i. 10U / mL recombinant human IL-2 for CD8 T cells ii. 100 U / mL recombinant human IL-2 and 25 U / ml recombinant human IL-15 for NK cells iii. 25ng / mL recombinant human GM-CSF for macrophages c. 1 x 10 cells 5 / mL x 100uL / well (1 x 10 4 / well) and incubated overnight at 37°C / 5% CO2. B. Nanoparticle and control conditions a. siRNA alone, untreated, and Lipofectamine + siRNA b. Standard LNP, GD3 3% LNP, GD3 5% LNP, PEG-less GD3 5% LNP, PSA DOTAP lipoplex C. Treatment and Dosage a. 12h and 48h processing time b. 30nM and 90nM siRNA c. Prepare NP suspension at 2x the desired final concentration in the above medium and add 100uL per well. d. N=3 experimental replicates, 2 technical replicates D. qPCR method a. GAPDH and ActinB expression levels were measured using β-actin as a reference gene to assess GAPDH knockdown. b. Two-step qPCR method E. Analysis a. Data normalized to siRNA treatment alone b. Relative fold change: 2^(-Δ ΔC T ) F. Results a. Table summarizing the mean relative fold change in GAPDH gene expression in each cell type. [Table 1]

[0102] While the present invention has been particularly shown and described with respect to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention encompassed by the appended claims.

Claims

1. A lipid nanoparticle composition comprising optionally nucleic acids and at least one ganglioside, wherein the lipid nanoparticles have an average particle size of 10 to 800 nm, preferably 30 to 200 nm.

2. The composition according to claim 1, wherein the lipid nanoparticles comprise cationic or ionizable lipids, and optionally phospholipids, cholesterol, and PEG lipids.

3. The composition according to claim 1, wherein the molar percentage of gangliosides in the total lipid composition is 0.1 to 50%, 0.5 to 20%, or 1 to 10%.

4. The composition according to claim 2, wherein the ganglioside comprises 1, 2, 3, or 4 sialic acid units.

5. The composition according to claim 2, comprising more than four ganglioside sialic acid units.

6. The composition according to claim 1, wherein lipid nanoparticles enhance cell transfection and targeting.

7. The composition according to claim 6, wherein the cells include immune cells and / or tumor cells.

8. The composition according to claim 7, wherein the cells are immune cells.

9. The composition according to claim 8, wherein the cells are T cells, NK cells, and macrophages.

10. The composition according to claim 9, wherein the cells are NK cells.

11. The composition according to claim 9, wherein the cell is a macrophage.

12. The composition according to claim 1, wherein the nucleic acid is DNA, MRA, siRNA, microRNA or aptamer, or a gene editing agent such as CRISPR-Cas9.

13. The composition according to claim 1 for administering nucleic acids to a subject who requires the administration of nucleic acids.

14. A composition according to claim 1 for the treatment of a disease or disorder in a subject who requires treatment for a disease or disorder.

15. The composition according to claim 14, wherein the disease is cancer.

16. The composition according to claim 13, wherein the nucleic acid is an anticancer agent or an immunotherapy agent.

17. The composition according to claim 14, wherein the disease is an autoimmune disease.

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