Novel lipid nanoparticle compositions and uses thereof for treating diseases in bone marrow

Abstract

The present invention provides a pharmaceutical-acceptable lipid nanoparticle (LNP) composition for delivering active agents to the bone marrow. The LNP comprises a ganglioside, a cholesterol, a phospholipid, and a cationic or ionizable lipid and exhibits superior transfection efficiency in bone marrow cells relative to other lipid nanoparticles that lack the ganglioside.

Description

[0001] NOVEL LIPID NANOPARTICLE COMPOSITIONS AND USES THEREOF FOR TREATING DISEASES IN BONE MARROW

[0002] RELATED APPLICATION

[0003] This application claims the benefit of U.S. Provisional Application No. 63 / 681,699, filed on August 9, 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), have 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 cargo.

[0006] LNP technology presents great opportunities to treat diseases such as sickle cell disease, thalassemia, chronic granulomatous disease, leukemia, and lymphoma by delivering nucleic acid cargos to the bone marrow and hematopoietic stem cells (HSCs). However, conventional LNPs cannot efficiently deliver nucleic acid cargo to the bone marrow and HSCs.

[0007] Therefore, there is an unmet need to develop a novel lipid nanoparticle system that can load various types of nucleic acids and deliver them to the bone marrow and HSCs.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention is based on the surprising discovery that a lipid nanoparticle (LNP) bearing sialic acid residues on the surface can deliver nucleic acid cargo to the bone marrow and the HSCs. The present invention provides an LNP composition comprising a lipid bearing, or substituted by, a sialic acid (SA) residue such as a ganglioside, a ganglioside derivative or mimetic, an SA-bearing entity, or a combination thereof. The lipid nanoparticle can incorporate nucleic acid molecules, or cargo, and deliver the cargo to the bone marrow via systemic dosing. 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 treating sickle cell disease, thalassemia, chronic granulomatous disease, leukemia, and lymphoma.

[0010] Sialic acid (SA), 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 (Neu5Ac 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.

[0011] 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 overall homeostasis.

[0012] 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) is 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.

[0013] Gangliosides are molecules composed of glycosphingolipids with one or more sialic acids linked to 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 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 at, for example, Avanti Polar Lipids, Birmingham, AL.

[0014] 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 in the present invention.

[0015] 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.

[0016] Typically, the lipid nanoparticles further comprise one or more chemical entities to form the lipid nanoparticles of the present invention. Preferably the lipid nanoparticles further comprise one or more cholesterol or cholesterol derivatives, phospholipids, cationic lipids, and ionizable lipids. The lipid nanoparticle can optionally comprise a PEG-lipid.

[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 lipids, DOTMA, ethyl PC’s, DDAB, pH-sensitive lipids, dioleoyl-3- trimethylammonium propane (DOTAP), DC-cholesterol, and GL67.

[0018] An ionizable lipid is 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, DODMA, and DODAP.

[0019] PEG-lipids include a class of 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 be preferably three (3) or more. For example, PEG(1000) represents a PEG unit with a 1000 MW 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-lipids can also be functionalized for bioconjugation. In one particular embodiment, the LNP of the present invention comprises a ganglioside, an ionizable lipid, cholesterol, 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 to adjust its overall performance properties.

[0020] The invention further relates to methods for the treatment of diseases or disorders, including but not limited to sickle cell disease, thalassemia, chronic granulomatous disease, leukemia, and lymphoma, in a subject in need thereof comprising administering to the subject the composition of the invention.

[0021] FIGURE LEGENDS

[0022] Figure 1. Overall Biodistribution of Cytofinity™ LNPs Compared with Standard LNPs.

[0023] Figure 2. Fluorescent Intensities 4 Hours after IV Dosing of Cytofinity™ and Standard LNPs Loaded with Cy5-GFP-mRNA.

[0024] Figure 3. tdTomato Expression in Femurs (3 A) and Tibias (3B).

[0025] Figure 4. Transfection of CD34+ Cells (Flow Cytometry).

[0026] Figure 5. Percentages of transfected long-term HSC (LSK CD150+CD48-) by traditional LNP (Example 11) and Cytofinity LNP (Example 14) in the mouse bone marrow determined by Flow Cytometry at 48-hour post-dosing. Cytofinity LNP is significantly more efficient than traditional LNP in targeting long-term HSCs in the bone marrow.

[0027] Figure 6. Percentages of transfected CD34+ cells by the traditional LNP (Std-LNP of Example 11), Cytofinityl LNP (Example 13) and Cytofinity2 LNP (Example 14) in the mouse bone marrow determined by Flow Cytometry at 48-hour post-dosing; Cytofinity LNPs are significantly more efficient than traditional LNPs in targeting CD34+ cells in the bone marrow.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Definitions

[0030] 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.

[0031] 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.

[0032] 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.

[0033] The term “particle” encompasses both nanoparticles and microparticles.

[0034] As used herein, “a” or “an” means one or more unless otherwise specified.

[0035] As used herein, “about” generally means up to ±10% of the particular term being modified.

[0036] The terms “sialic acid residue” and “sialic acid moiety” as well as their plural referents, and the like, are used interchangeably herein.

[0037] 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.

[0038] “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, 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.

[0039] Ganglioside

[0040] 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.

[0041] Ganglioside GM1

[0042]

[0043] Ganglioside GD3 Disodium Salt

[0044] 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 gangliosides GM1, GM2 and GD3 are shown above.

[0045] 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. 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.

[0046] 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.

[0047] The gangliosides can be added to the formulation at least 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 0.5-50%, 1-20%, 2-15%, 3-10% or about 3%.

[0048] Cholesterol

[0049] 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:

[0050] 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.

[0051] Cholesterol derivatives also include vitamin D and other open ring derivatives including cholecalciferol and ergocalciferol:

[0052] And compounds having the structure: 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.

[0053] 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.

[0054] Phospholipid

[0055] The lipid nanoparticles preferably further comprise one or more phospholipids.

[0056] 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).

[0057] 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.

[0058] In a preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC).

[0059] 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 phospholipid's molar ratio is preferably between 1-50%, 2-20%, 3-15%, 5-15%, or about 10% of the total lipid composition.

[0060] PEG lipid

[0061] 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, 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 any inflammatory reactions and maintain homeostasis.

[0062] Cationic and Ionizable Lipid

[0063] Thus, a lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino) lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0064] In a particular embodiment, the 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 or ionizable (amino) 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.

[0065] 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), N 1 -[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-[ 1,3]- di oxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen- 19-yl 4-(dimethylamino)butanoate (DLin- MC3- DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)- [l,3]-dioxolane (DLin-KC2-DMA),

[0066] 1.2- dioleyloxy-N,N-dimethylaminopropane (DODMA), 2-({8-[(3.beta.)-cholest-5-en- 3- yloxy]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)-

[0067] 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-l- yloxy]propan-l- amine (Octyl-CLinDMA (2S)).

[0068] 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. Ionizable lipids can also form the complex with the SA-containing entity while promoting endosome escape and reducing toxicity. Examples of commercially available ionizable lipids include DLin- KC2-DMA, DLin-MC3-DMA, DLin-DMA, LP-01, DODMA, DODAP, ALC- 0315, SM-102, SS-OP, SS-EC, etc.

[0069] The ionizable lipid can be added to the formulation in an amount of at least about

[0070] O.1% molar weight percentage of total solids in the nanoparticle composition. The molar ratio of the cationic or ionizable lipid can preferably be between 1-50%, 10-45%, or, more preferably, between 15 and 40% of the total lipid composition.

[0071] Active Agent

[0072] The anti-inflammatory 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.

[0073] The concentration of nucleic acids to LNPs can be characterized by an 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.

[0074] 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.

[0075] Production of the Particles

[0076] The lipid nanoparticle described herein can be manufactured by a coprecipitation process. In general, the ganglioside can be dissolved separately or along with other lipids (such as ionizable lipid, DSPC and cholesterol) in a preferably water-miscible organic solvent, such as 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.

[0077] The particles may also be manufactured using a pre-assembled device such as a T- mixer, or an automated, microfluidic device such as NanoAssemblr™ (Ignite, Blaze, etc.) of Precision Nanosystems, Automated Nanoparticle System of Particle Works, and iNano L series of Micro&Nano.

[0078] Exemplary solvents miscible with water include methanol, ethanol, acetone, tetrahydrofuran (THF), acetonitrile, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF).

[0079] The 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.

[0080] 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.

[0081] Such obtained nanoparticle suspension (either by co-precipitation or emulsification) can be further purified with dialysis, centrifugation, or tangential flow.

[0082] Particle Sizes

[0083] The size of the subject nanoparticles is from about 1 nm to about 2 pm, preferably from about 10 nm to about 1 pm, and most preferably from about 20 nm to about 500 nm. For example, the nanoparticles may have an average size between about 50 and 900 nm, such as about 50, 75, 100, 300, 500, 700, or 900 nm.

[0084] 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.

[0085] Pharmaceutical Composition

[0086] 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 or inclusion in a pharmaceutical composition with a compound of this invention 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. It will also be appreciated that certain of the subject particles of the present invention can exist in free form for treatment or, where appropriate, as a pharmaceutically acceptable derivative thereof.

[0087] Preferably, the pharmaceutical compositions of the present invention additionally comprise a pharmaceutically acceptable carrier, which, as used herein, includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, 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.

[0088] Some examples of materials which can 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 carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants, adjuvants including but not limited to Alum, MF59, AS04, and CPG can also be present in the composition, according to the judgment of the formulator.

[0089] 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).

[0090] In one embodiment, the method includes administering the subject composition or pharmaceutical composition comprising the subject lipid nanoparticles by any suitable means or routes, such as orally, nasally, intraperitoneally, intravitreally, intravenously, intramuscularly, intracranially, intrathecally, transdermally, or subcutaneously. In a particular embodiment, the particles are administered via an intravenous route.

[0091] Exemplary Uses

[0092] The lipid nanoparticles and compositions thereof have numerous applications including in therapeutic methods. 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.

[0093] The lipid nanoparticles described herein can be used to treat various diseases, including but not limited to sickle cell disease, thalassemia, chronic granulomatous disease, leukemia, and lymphoma.

[0094] EXEMPLIFICATION

[0095] Example 1. Preparation of traditional lipid nanoparticles comprising a Cy5-labeled Green Fluorescence (GFP) mRNA

[0096] Stock solutions of the ionizable lipid DLin-MC3-DMA (MC3), cholesterol (Choi), and l,2-distearoyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) at 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 63.74 pL MC3 solution, 29.55 pL Choi solution, 15.69 pL DSPC solution, and 14.94 pL PEG-lipid solution; the volume was brought up to 1.2 mL with ethanol, and the lipid mixture was vortexed. A 10 mM citrate buffer (pH 3) solution 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, 100 uL of EZ Cap™ Cy5 EGFP mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0097] The lipid and cargo packets were loaded into 10 mL Luer-lok 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; aqueous flow rate = 24 mL / min, lipid flow rate = 8 mL / min. Lipid nanoparticles were collected from the T-mixer in a 50 mL conical tube. The LNP suspension was transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 20K MWCO and dialyzed against phosphate-buffered saline (PBS) for 6 h to remove the alcohol. The solution was concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution.

[0098] Example 2. Preparation of traditional lipid nanoparticles comprising Cre-mRNA DLin-MC3-DMA, cholesterol, DSPC, and DMG-PEG 2000 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, DSPC, and DMG-PEG 2000 were dissolved in appropriate volumes of ethyl alcohol to prepare solutions at concentrations of 50, 20, 20, and 10 mg / mL, respectively. 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 prepare a 10 mM solution of citrate buffer (pH 3). A 0.0283 mg / mL solution of CleanCap® Cre mRNA (5moU) was made by adding 50.4 uL of 1 mg / mL mRNA solution to 1.727 mL of 10 mM citrate buffer.

[0099] DLin-MC3-DMA, cholesterol, DSPC, and DMG-PEG 2000 were combined at a molar ratio of 50:38.5: 10: 1.5 for an N:P ratio of 6 and a total lipid concentration of 3 mM by adding 12, 13.9, 7.4, and 3.5 uL of the stocks, respectively, to 587 uL ethanol. The lipid and mRNA solutions were loaded into 1 mL and 3 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 :3 flow rate ratio, total volume 2.3 mL, and 0.2 mL start waste. The mixed nanoparticles were collected, diluted, and washed in IX PBS to reduce the organic solvent concentration. The nanoparticle suspension was transferred to an Amicon Ultra centrifugal filter with 100 kDa molecular weight cut-off and spun at 2000 RCF to a volume of 0.5 mL. Aliquots were stored with 15% w / v sucrose at -80 °C.

[0100] Example 3. Preparation of Cytofmity™ LNP comprising a Cy5-labeled GFP-mRNA

[0101] MC3, Choi, and DSPC stock solutions were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A 10 mg / mL stock solution of PEG-lipid was prepared by dissolving the proper amount of weighed lipid in ethanol. A 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 63.74 pL MC3 solution, 29.37 pL Choi solution, 14.41 pL DSPC solution, 15.25 pL PEG-lipid solution, and 18.32 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM citrate buffer (pH 3) solution 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, 100 uL of EZ Cap™ Cy5 EGFP mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0102] The lipid and cargo packets were loaded into 10 mL Luer-lok 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; cargodipid flow rate ratio (FRR) = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. To remove alcohol, the collected LNP suspension was transferred into a pre-hydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS overnight. The solution was concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at - 20° C in a 5% w / v sucrose solution.

[0103] Example 4. Preparation of Cytofinity™ LNP comprising a Cy5-labeled GFP-mRNA

[0104] MC3, Choi, and DSPC stock solutions were prepared by dissolving the appropriate amount of weighed lipids in ethanol for 20 mg / mL concentrations. A 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 63.74 pL MC3 solution, 28.98 pL Choi solution, 14.41 pL DSPC solution, and 30.53 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, 100 uL of EZ Cap™ Cy5 EGFP mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0105] The lipid and cargo packets were loaded into 10 mL Luer-lok 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; flow rate ratio (FRR) = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. To remove alcohol, the collected LNP suspension was transferred into a prehydrated Slide-A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS overnight. The solution was concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution. Example 5. Preparation of Cytofmity™ LNP comprising a Cy5-labeled Green Fluorescence Protein (GFP) mRNA

[0106] MC3, Choi, and DSPC stock solutions 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 63.74 pL MC3 solution, 29.2 pL Choi solution, 15.35 pL DSPC solution, and 65.03 pL GD3 solution; the volume was brought up to 1.2 mL with ethanol and the lipid mixture was vortexed. A 10 mM citrate buffer (pH 3) solution 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, 100 uL of EZ Cap™ Cy5 EGFP mRNA solution (1 mg / mL) was diluted to 3.2 mL in 10 mM citrate buffer.

[0107] The lipid and cargo packets were loaded into 10 mL Luer-lok 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; flow rate ratio (FRR) = 3: 1; cargo flow rate = 24 mL / min, lipid flow rate = 8 mL / min. To remove alcohol, the collected LNP suspension was transferred into a prehydrated Slide- A-Lyzer™ dialysis cassette with a 10K MWCO and dialyzed against PBS overnight. The solution was concentrated at 2000 RCF in an Amicon® Ultra centrifugal filter unit with a 100K MWCO. The sample was collected from the upper filter and stored at -20° C in a 5% w / v sucrose solution.

[0108] Example 6. Preparation of Cytofmity™ LNP comprising Cre-mRNA

[0109] GD3 (bovine buttermilk) (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 GD3 was prepared in methanol. Lipid solutions were warmed at 40 °C to ensure full solubilization before preparing the lipid packet. UltraPure™ DNase / RNase-Free distilled water was used to prepare a 10 mM solution of citrate buffer (pH 3). A 0.0196 mg / mL solution of CleanCap® Cre mRNA (5moU) was made by adding 50.4 uL of 1 mg / mL mRNA solution to 2.525 mL of 10 mM citrate buffer.

[0110] 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 3 mM by adding 21.4, 12, 14.7, and 10.4 uL of the stocks, respectively, to 619 uL ethanol. The lipid and mRNA solutions were loaded into 1 mL and 3 mL BD Luer-Lok syringes and subjected to microfluidic mixing with the iNano L+ using an S-SDM cartridge. Instrument settings were the following: 20 mL / min total flow rate, 1 :4 flow rate ratio, total volume 3.2 mL, and 0.2 mL start waste. The mixed nanoparticles were collected, diluted, and washed in IX PBS to reduce the organic solvent concentration. The nanoparticle suspension was transferred to an Amicon Ultra centrifugal filter with 100 kDa molecular weight cut-off and spun at 2000 RCF to a volume of 0.4 mL. Aliquots were stored with 15% w / v sucrose at -80 °C.

[0111] Example 7. Preparation of Cytofinity™ LNP comprising Cre-mRNA

[0112] GD3 (bovine buttermilk) (ammonium salt), ALC-0315, 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. ALC-0315, 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. Lipid solutions were warmed at 40 °C to ensure full solubilization before preparing the lipid packet. UltraPure™ DNase / RNase-Free distilled water was used to prepare a 10 mM solution of citrate buffer (pH 3). A 0.0204 mg / mL solution of CleanCap® Cre mRNA (5moU) was made by adding 100.7 uL of 1 mg / mL mRNA solution to 4.798 mL of 10 mM citrate buffer. 37.2 uL of the 5 mg / mL GD3 was added to this mRNA solution.

[0113] Taking into account the GD3 in the aqueous solution, GD3, ALC-0315, cholesterol, and DSPC were combined to achieve a molar ratio of 5:48:35: 12 for an N:P ratio of 8 and a total lipid concentration of 4 mM by adding 39.1, 38.3, 35.2, and 24.6 uL of the stocks, respectively, to 1162 uL ethanol. The lipid and mRNA solutions were loaded into 3 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 5.9 mL, and 0.3 mL start waste. The mixed nanoparticles were collected and then diluted and washed in IX PBS to reduce concentration of organic solvent. The nanoparticle suspension was transferred to an Amicon Ultra centrifugal filter with 100 kDa molecular weight cut-off and spun at 2000 RCF to a volume of 0.45 mL. Aliquots were stored with 15% w / v sucrose at -80 °C. Example 8. IVIS imaging on bone marrow

[0114] A biodistribution study was conducted with BALB / c mice (n=3). LNPs loaded with Cy5 EGFP mRNA were administered to the mice via tail-vein injection at a dose of 10 pg mRNA per mouse, or approximately 0.5 mg RNA / kg. 4 hours after the dosing, all mice were sacrificed, and the left femur of each animal was collected and imaged with IVIS imaging. Example 9. Biodistribution study

[0115] Male Ail4 mice (B6.Cg-Gt(ROSA) 26Sortml4 (CAG-TdTomato) Hze / J, Strain #007914) that were 8-10 weeks of age on arrival were acclimated for at least 72 h. These mice have a loxP-flanked STOP cassette preventing transcription of a CAG promoter-driven tdTomato and upon Cre-mediated recombination express tdTomato fluorescence. The mice were provided ad libitum Envigo irradiated 2918 diet and filtered tap water. Clinical observations and body weights were recorded throughout the experiment. Three animals were included in each treatment group.

[0116] Cre mRNA-loaded lipid nanoparticle test materials were thawed, and suspensions in saline were prepared at 46.1 pg RNA / mL. Each animal was dosed via a single tail vein intravenous injection with a volume of 220 pL, equating to 10 pg Cre-mRNA encapsulated in the LNPs.

[0117] Six days following dosing, animals were anesthetized via isoflurane for in-life wholebody IVIS imaging to visualize and quantify the TdTomato fluorescent signal. Next, the mice were euthanized by CO2 asphyxiation, and exsanguination was performed: the maximum obtainable volume of whole blood was collected into EDTA tubes and stored chilled. Intracardiac perfusion with 30 mL cold PBS was done, setting the needle into the left ventricle lumen with the right atrium incised as a drainage outlet. The brain, heart, lungs, liver, spleen, kidneys, femurs, tibias, quadriceps muscles were collected and imaged per group with the IVIS.

[0118] All organs except the bones were fixed in 4% PFA for 24 h. Then, brains were stored in PBS, and all other organs were stored in 70% ethanol. The tibia and femur bones were placed into tubes with PBS and kept chilled.

[0119] Example 10. Flow cytometry analysis of bone marrow

[0120] To isolate the bone marrow from the Ail4 mice, each animal’s tibia and femur bones were flushed with IX PBS supplemented with 5 mM EDTA and 1% fetal calf serum onto a strainer, using a flat syringe top to break up and push the marrow through. Next, the cells were centrifuged in PBS for 5 minutes at 350 RCF. Cells were resuspended in 1 mL PBS and counted before plating at 2e6 cells / well in a 96 well V-bottom plate.

[0121] First, the cells were incubated on ice with APC-eFluor 780 fixable viability dye / mouse Fc block reagent for 20 minutes, then centrifuged at 350 RCF for 5 minutes before discarding the supernatant. Cells were incubated on ice for 40 minutes with a surface-staining antibody cocktail in BD stain buffer (see panel design in the table below). Again, cells were centrifuged, and the supernatant was discarded. After fixation, the cells were resuspended in 300 pL BD stain buffer. Unstained control, viability control, and fluorescence minus one (FMO) control were included.

[0122] A Thermo Fisher Attune NxT with four lasers and an autosampler was used to perform flow cytometry on the cells. Suspended cell samples were recorded at 100 pL / min with a 270 pL acquisition volume. Settings were such that there were 7 tip rinsing cycles and 7 sample mixing cycles between injections.

[0123] The following flow cytometry gating strategy was used to identify HSCs expressing tdTomato, indicative of successful HSC transfection. All recorded cells were gated for single cells, which were gated for live cells and CD34+ cells, of which the tdTomato+ population was quantified.

[0124] Example 11. Preparation of traditional lipid nanoparticles (LNP) comprising a Green Fluorescence Protein (GFP) mRNA using ALC-0315 as the ionizable lipid

[0125] Stock solutions of the ionizable lipid ALC-0315 (ALC) at 50 mg / mL, cholesterol (Choi), and l,2-distearoyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) at 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 adding 72.1 pL ALC solution, 70.1 pL Choi solution, 37.2 pL DSPC solution, 35.4 pL PEG-lipid solution to 452.0 pL ethanol, and the lipid mixture was vortexed and heated to ensure the formation of a clear solution. A 10 mM citrate buffer (pH 3) solution was prepared with UltraPure RNAse / DNAse free water. The cargo packet was prepared at 0.1 mg / mL by adding 70.6 uL of 2.833 mg / mL GFP-mRNA stock to 1.93 mL citrate buffer solution. Lipid and cargo packets were prepared at 5% excess.

[0126] The lipid mix was loaded into a 1-mL Luer-lock syringe, and RNA solution into a 3 mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with an S-SDM cartridge and the following settings: TFR = 20 mL / min; FRR = 3 (cargo): 1 (lipid). Lipid nanoparticles were collected from the iNano L+ mixer and diluted with 2 mL pH 7.4 phosphate-buffered saline (PBS). The LNP suspension was transferred into a 3-mL Slide- A-Lyzer™ dialysis cassette with 20K MWCO and dialyzed against PBS for 6 hours. The solution was concentrated in a 100K MWCO Amicon Ultra-15 centrifugal filter to a volume of 975 uL. The sample was collected from the upper filter and stored at -80° C in a 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 75.0 nm, a PDI of 0.162, a zeta potential of -1.0 mV, and an RNA encapsulation efficiency (EE) of 86.3%.

[0127] Example 12. Preparation of traditional lipid nanoparticles comprising a Green Fluorescence (GFP) mRNA using Al 1T2 as the ionizable lipid

[0128] Stock solutions of the ionizable lipid Al 1T2 (Al 1), cholesterol (Choi), and 1,2- distearoyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) at 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 ethanol. The lipid packet was made by adding 166.9 pL Al 1 solution, 64.5 pL Choi solution, 34.2 pL DSPC solution, 32.6 pL PEG-lipid solution to 316.0 pL ethanol, and the lipid mixture was vortexed and heated to ensure the formation of a clear solution. A 10 mM citrate buffer (pH 3) solution was prepared with UltraPure RNAse / DNAse free water. The cargo packet was prepared at 0.1 mg / mL by adding 65.0 pL of 2.833 mg / mL GFP-mRNA stock to 1.78 mL citrate buffer solution.

[0129] The lipid mix was loaded into a 1-mL Luer-lock syringe, and RNA solution into a 3 mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 20 mL / min; FRR = 3 (cargo): 1 (lipid). Total volume = 2.4 mL; cargo = 1.8 mL; lipids = 0.6 mL; start waste 0.2 mL. Lipid nanoparticles were collected from the iNanoL+ mixer, transferred to a Slide-A-Lyzer dialysis cassette with 20K MWCO, dialyzed against lx PBS for 4 hours, and diluted with pH 7.4 phosphate-buffered saline (PBS) to a total volume of 15 mL. The LNP suspension was concentrated in 100K MWCO Amicon Ultra- 15 centrifugal filter to a volume of 0.9 mL, followed by filtration with a 0.22 mm syringe filter. The LNP sample was stored at -80°C in a 15% w / v sucrose solution. The resulting LNP was found to have a Z-average particle size of 79.1 nm, a PDI of 0.196, a zeta potential of -0.25 mV, and an RNA encapsulation efficiency (EE) of 93.6%.

[0130] Example 13. Preparation of Cytofmity™ LNP comprising a GFP-mRNA and 7% ganglioside GM1 using ALC-0315 as the ionizable lipid

[0131] Stock solutions of the ionizable lipid ALC-0315 (ALC) at 50 mg / mL, cholesterol (Choi), and l,2-distearoyl-sn-glycero-3-phosphocholine 18:0 PC (DSPC) at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A stock solution of ganglioside GM1 at 10 mg / mL was prepared by dissolving a proper amount of GM1 in methanol. The lipid packet was made by adding 72.1 pL ALC solution, 63.9 pL Choi solution, 51.4 pL DSPC solution, 109.6 pL GM1 solution to 704.0 pL ethanol, and the lipid mixture was vortexed and heated to ensure the formation of a clear solution. A 10 mM citrate buffer (pH 3) solution was prepared with UltraPure RNAse / DNAse free water. The cargo packet was prepared at 50 pg / mL by adding 70.6 uL of 2.833 mg / mL GFP-mRNA stock to 3.94 mL citrate buffer solution. Lipid and cargo packets were prepared at 5% excess.

[0132] The lipid mix was loaded into a 1-mL Luer-lock syringe and RNA solution into a 5 mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with an S-SDM cartridge and the following settings: TFR = 20 mL / min; FRR = 4 (cargo): 1 (lipid). Lipid nanoparticles were collected from the iNanoL+ mixer and diluted 1 :4 with 20 mL pH 7.4 phosphate-buffered saline (PBS). The LNP suspension was transferred into a 30- mL Slide-A-Lyzer™ dialysis cassette with 20K MWCO and dialyzed against PBS for 6 hours. The solution was concentrated in a 100K MWCO Amicon Ultra- 15 centrifugal filter to a volume of 700 uL. The sample was collected from the upper filter, filtered through a 0.22 pm syringe filter, and stored at -80° C in a 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 65.1 nm, a PDI of 0.04, a zeta potential of - 9.8 mV, and an RNA encapsulation efficiency (EE) of 90.7%. Example 14. Preparation of Cytofmity™ LNP comprising a GFP-mRNA and 7% ganglioside GM1 using Al 1T2 as the ionizable lipid

[0133] Stock solutions of Al 1, cholesterol (Choi), and DSPC at 20 mg / mL were prepared by dissolving the appropriate amount of weighed lipids in ethanol. A stock solution of ganglioside GM1 at 10 mg / mL was prepared by dissolving a proper amount of GM1 in methanol. The lipid packet was made by adding 181.2 pL Al 1 solution, 63.9 pL Choi solution, 51.4 pL DSPC solution, 109.6 pL GM1 solution to 595.0 pL ethanol. The lipid mixtures were vortexed and heated at 40°C to ensure complete solubilization. A 10 mM citrate buffer (pH 3) solution was prepared with UltraPure RNAse / DNAse free water. The cargo packet was prepared at 50 pg / mL by adding 70.6 pL of 2.833 mg / mL eGFP-mRNA stock to 3.94 mL citrate buffer solution.

[0134] The lipid mix was loaded into a 1-mL Luer-lock syringe and RNA solution into a 5- mL Luer-lock syringe. The two syringes were mounted on an iNano L+ microfluidic mixer with the following setting: TFR = 20 mL / min; FRR = 4 (cargo): 1 (lipid). Total volume = 4.8 mL; cargo = 3.84 mL; lipid = 0.96 mL; start waste = 0.2 mL. LNPs were collected from the iNanoL+ mixer and diluted with pH 7.4 PBS to 20 mL, transferred into a Slide-A-Lyzer™ dialysis cassette with 20K MWCO, dialyzed against PBS for 6 hours, concentrated in a 100K MWCO Amicon Ultra-15 centrifugal filter unit, followed by filtration through a 0.22 pm syringe filter. The LNP sample was collected and stored at -80° C in 15% w / v sucrose solution. The resulting lipid nanoparticles were found to have a Z-average particle size of 64.8 nm, a PDI of 0.06, a zeta potential of -11.1 mV, and an RNA encapsulation efficiency (EE) of 98.4%.

[0135] Example 15. In vivo experiment evaluating the transfection of bone marrow stem cells after systemic administration of LNPs.

[0136] BALB / c (000651) female mice (6 weeks old, ~20 g) were acclimated to the test facility for 4 days and fed the 2920X diet. LNPs were thawed at ambient temperature. For the Cytofmity 1 LNP (Example 13), which had an encapsulated mRNA concentration of 154 pg / mL, 649 pL of the LNP solution was diluted with 751 pL PBS. For the Cytofinity2 LNP (Example 14), which had an encapsulated mRNA concentration of 146.6 pg / mL, 682 pL of the LNP solution was diluted with 718 pL PBS. For the traditional LNP (Example 1), which had an encapsulated mRNA concentration of 105.6 pg / mL, 947 pL of the LNP solution was diluted with 453 pL PBS. LNP suspensions were loaded into insulin syringes (U-100 BD Micro-Fine IV Single Unit Graduation, 28 G, 0.5 in, 1 mL) and 140 pL (~7 mL / kg) of the respective LNP solution was injected into the retro-orbital venous plexus of each mouse (n=8) while the mouse was under anesthesia with a single 10 pg dose.

[0137] 24 hours after dosing, 4 out of the 8 mice in each test group were sacrificed. No significant changes in body weight or adverse health conditions were observed. Mice were euthanized with CO2, and the whole blood of each mouse was collected in K2EDTA tubes. Intracardiac perfusion was performed on each mouse. The tibia and femur of each of the 4 mice were collected by dissecting and separating the hind leg muscle and kept on ice in RPMI-1640 medium. Other organs of interest, including the liver, lung, and spleen, were also collected. Ex vivo imaging of the GFP signal on each tissue was performed with an IVIS instrument.

[0138] 48 hours after dosing, the remaining 4 mice in each test group were sacrificed. No significant changes in body weight or adverse health conditions were observed. Mice were euthanized with CO2, and the whole blood of each mouse was collected in K2EDTA tubes. Intracardiac perfusion was performed on each mouse. The tibia and femur of each of the 4 mice were collected by dissecting and separating the hind leg muscle and kept on ice in RPMI-1640 medium. Other organs of interest, including the liver, lung, and spleen, were also collected. Ex vivo imaging of the GFP signal on each tissue was performed with an IVIS instrument.

[0139] Traditional LNPs generally showed higher GFP expression in the liver than Cytofinity LNPs. For example, the traditional LNP of Example 1 and 2 both had stronger GFP fluorescence in the liver than the Cytofinity LNP of Example 10. As indicated by Figure 5, the Cytofinity™ LNP of Example 10 had much less GFP expression in the liver than the traditional LNPs of Example 1 and 2 at the 48-hour post-dosing. This confirms that the Cytofinity™ LNP of the present invention has the advantage of reducing LNP -mediated liver toxicity.

[0140] The bones were placed in phosphate-buffered saline (PBS) on a petri dish for the isolation of bone marrow cells. Both ends of the tibias and femurs were cut. The bones were then placed into 0.6 mL tubes that had a hole made in the bottom with an 18G needle. These tubes were fitted into 1.5 mL microcentrifuge tubes and centrifuged at 6000 rpm for 1 minute. The resulting cell pellet in the 1.5 mL tube was suspended in 1 mL of RBC lysis buffer and incubated at room temperature (RT) for 5 minutes. For each sample, a 5 mL round-bottom tube equipped with a cell strainer cap was rinsed with ice-cold PBS. The cell solution was poured through the strainer, and an additional 1 mL of PBS was used to rinse the filter. The tubes were centrifuged at 1500 rpm for 5 minutes, and then the cell pellet was resuspended in 1 mL of PBS and centrifuged again.

[0141] Approximately 10 million cells were used for staining. The cell pellet was resuspended in 50 pL of the Live / Dead Fixable Near-IR dead cell stain kit (APC-Cy7, 1 : 1000 dilution) (Invitrogen) and incubated at 4 °C for 15 minutes. Following incubation, the cells were washed with 1 mL of FACS buffer and then blocked with 50 pL of TruStain FcX™ PLUS anti-mouse CD16 / 32 antibody (BioLegend) at a dilution of 1 :50 for 10 minutes at 4 °C. An antibody cocktail master mix was prepared, and 50 pL of this mix was added to each sample. The antibody staining panel included the following antibodies: APC anti-mouse CD34 (1 :50 dilution, BioLegend), Brilliant Violet 421™ anti-mouse lineage cocktail (1 :25 dilution, BioLegend), PE / Cyanine7 anti-mouse Ly-6A / E (Sca-1) (1 :50 dilution, BioLegend), Brilliant Violet 711™ anti-mouse c-Kit (1 :50 dilution, BioLegend), Brilliant Violet 605™ anti-mouse CD48 (1 :50 dilution, BioLegend), PE anti-mouse SLAM (1 :50 dilution, BioLegend), and Alexa Fluor® 700 anti-mouse CD45 (1 :25 dilution, BioLegend). Single stain controls were prepared with beads to create the compensation matrix. After a 30-minute incubation at 4 °C, the stained cells were washed and resuspended in FACS buffer. The samples were then resuspended in 3.5 mL of FACS buffer and analyzed on the 4-laser Attune NxT flow cytometer (Thermo Fisher) using a 3 mL acquisition volume and a flow rate of 1000 pL / min. Unstained cells were used to adjust the forward scatter (FSC) and side scatter (SSC) voltages.

[0142] Flow cytometry data were analyzed using Flow Jo (BD) to identify hematopoietic stem cell (HSC) populations that expressed the GFP signal, an indication of successful transfection by the mRNA-loaded lipid nanoparticles (LNPs). Two gating strategies were applied: the first identified single cells, followed by live cells, then CD34+ cells, and finally GFP+ cells. The second strategy is gated for single cells, live cells, Lineage- / low, c-Kit+ / Sca- 1+, CD48- / CD150+, and GFP+ cells. The results demonstrated that the Cytofinity LNP of Example 14 significantly increased mRNA transfection in CD34+ bone marrow cells compared to the traditional LNPs (Example 11 and Example 12) delivery systems (see Figures 5 and 6).

[0143] The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.

[0144] 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. It will also be understood that none of the embodiments described herein are mutually exclusive and may be combined in various ways without departing from the scope of the invention encompassed by the appended claims.

Claims

CLAIMS1. A pharmaceutically acceptable lipid nanoparticle composition for the delivery of an active agent to bone marrow cells, wherein the lipid nanoparticle comprises a ganglioside, cholesterol, phospholipid, and a cationic or ionizable lipid.

2. The lipid nanoparticle of claim 1, wherein the ganglioside is selected from GM1, GM2, GM3, asialo-GMl, GAI, asialo-GM2, GA2, GDla, GDlb, GD2, GD3, GTla, GTlb, GTlc, OAc-GTlb, GT3, and GQ1.

3. The lipid nanoparticle of claim 1 or claim 2, wherein the ganglioside is GM1.

4. The lipid nanoparticle of any one of the preceding claims, wherein the molar ratio of the ganglioside to total lipids can be between 0.5-50%, 1-20%, 2- 15%, or 3- 10%.

5. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle has an average particle size of 5-1,000 nm, preferably 20-500 nm, or 30-200 nm.

6. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle comprises an ionizable lipid.

7. The lipid nanoparticle of any one of the preceding claims, wherein the ionizable lipid is the ionizable lipid is selected from DLin-KC2-DMA, DLin-MC3-DMA, DLin-DMA, ALC-0315, SM-102, DODMA, and DODAP.

8. The lipid nanoparticle of any one of the preceding claims, wherein the lipid nanoparticle further comprises an optional PEG-lipid.

9. The lipid nanoparticle of any one of the preceding claims, wherein the phospholipid is DSPC.

10. The lipid nanoparticle of any one of the preceding claims, wherein the active agent is a nucleic acid.

11. The lipid nanoparticle of any one of the preceding claims, wherein the active agent is encapsulated within the lipid nanoparticle.

12. The lipid nanoparticle of any one of the preceding claims, wherein the active agent is a DNA, an mRNA, a siRNA, a microRNA, an aptamer, or a gene-editing agent.

13. The lipid nanoparticle of any one of the preceding claims, wherein the composition exhibits higher transfection efficiency in bone marrow cells compared to a standard lipid nanoparticle composition that lacks a ganglioside, as determined by delivery and expression of a nucleic acid cargo in bone marrow cells in vivo or ex vivo.

14. The lipid nanoparticle of claim 13, wherein the bone marrow cell is a hematopoietic stem cell (HSC).

15. The lipid nanoparticle of claim 13 or claim 14, wherein higher transfection efficiency is indicated by a higher percentage of CD34+ cells that is transfected (preferably measured by flow cytometry analysis).

16. A pharmaceutically acceptable lipid nanoparticle composition for the delivery of an active agent to bone marrow cells, comprising a ganglioside, cholesterol, a phospholipid, and a cationic or ionizable lipid, wherein the presence of the ganglioside enhances delivery of the active agent to bone marrow cells relative to a lipid nanoparticle lacking the ganglioside.

17. A method of transfecting bone marrow cells in a subject, comprising administering to the subject an effective amount of the lipid nanoparticle composition of any one of the preceding claims.

18. The method of claim 17, wherein transfection is indicated by expression of the nucleic acid in the cell.

19. The method of claim 17 or claim 18, wherein the lipid nanoparticle composition exhibits higher transfection efficiency in bone marrow cells compared to a lipid nanoparticle lacking the ganglioside.

20. A method of delivering an active agent to bone marrow cells in a subject, comprising systemically administering the lipid nanoparticle composition of any one of claims 1-16, wherein the lipid nanoparticle encapsulates the active agent.

21. The method of claim 20, wherein the lipid nanoparticle preferentially delivers the therapeutic nucleic acid to bone marrow cells relative to liver cells, as determined by measuring reporter gene expression, fluorescent signal, or nucleic acid levels in bone marrow and liver tissues following systemic administration, wherein the bone marrow-to-liver signal ratio is greater than that observed with a lipid nanoparticle composition lacking the ganglioside.

22. A method of treating a disease or condition involving bone marrow or hematopoietic dysfunction in a subject, comprising administering to the subject the lipid nanoparticle composition of any one of claims 1-16, wherein the lipid nanoparticle encapsulates a therapeutic nucleic acid.

23. The method of claim 22, wherein the disease or condition is selected from sickle cell disease, thalassemia, chronic granulomatous disease, hematologic malignancies, bone marrow failure syndromes, genetic blood disorders, and immune deficiencies.

24. The method of claim 22, wherein the disease or condition is a cancer.

25. The method of claim 24, wherein the cancer is selected from leukemia, lymphoma, multiple myeloma, or metastatic solid tumor (e.g., with bone marrow involvement).

26. The method of any one of claims 22-25, wherein the therapeutic nucleic acid is a gene-editing agent for correcting a genetic defect in hematopoietic stem cells.

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