Poly(alkyloxazoline)-lipid conjugates and lipid particles containing same

Abstract

The present disclosure provides poly(alkyloxazoline)-lipid conjugates, lipid particles comprising such conjugates, and methods of administering the lipid particles for nucleic acid delivery to a cell or a subject.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 305,211, filed January 31, 2022, the disclosure of which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] background Lipid nanoparticles (LNPs) have become a mainstream pharmaceutical modality with the advent of Onpattro® and mRNA-LNP COVID vaccines. LNPs typically have four different lipid components, including cationic lipids, non-cationic lipids, e.g., phospholipids, and sterols, and polymer-conjugated lipids. Each plays a specific role and is carefully assembled in specific ratios to provide a highly potent and well-tolerated delivery vehicle.

[0003] Cationic lipids are positively charged at acidic pH and promote the encapsulation of negatively charged nucleic acid (e.g., mRNA) payloads during particle formation. Following cellular uptake of the LNP, this further drives endosomal fusion and intracytoplasmic release of the payload. Phospholipids and cholesterol are often referred to as structural lipids, and concentrations are selected to optimize particle size, encapsulation, and stability. Polymer-conjugated lipids control particle size during formation and prevent particle aggregation by sterically stabilizing the LNP. It is on the surface of the particle, with the hydrophilic polymer facing outward and in contact with the aqueous environment, and the lipid component is buried in the particle to lock it into place.

[0004] Throughout the past decades of development of lipid particles for nucleic acid delivery, the polymer of choice for the polymer-conjugated lipid component has been poly(ethylene glycol) (PEG). PEG-lipids have been successfully used in several approved nucleic acid-containing LNPs, including the COVID-19 vaccines Comirnaty® and SpikeVax. However, due to exposure to PEG in other products (e.g., cosmetics), some populations are thought to have developed antibodies that recognize and bind to PEG, which may lead to hypersensitivity and loss of drug activity (Judge et al. Mol Ther. 2006, 13(2):328-37 (Non-Patent Document 1)). Shortly after the U.S. Food and Drug Administration (FDA) issued emergency use authorization for the COVID vaccines mentioned above, cases of anaphylactoid-type reactions after administration began to be reported (Shimabukuro et al. JAMA. 2021, 325(11):1101-1102 (Non-Patent Document 2)). Although the cause of these cases has not yet been proven, some have suggested that the cause may be the inclusion of PEG in these products. Thus, there is a need for alternative polymers that form effective particles with desirable activity and tolerability profiles. The present disclosure addresses this and other needs. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Judge et al. Mol Ther. 2006, 13(2):328-37 [Non-Patent Document 2] Shimabukuro et al. JAMA. 2021, 325(11):1101-1102 Summary of the Invention

[0006] Quick Overview Poly(alkyloxazoline)-lipid conjugates according to formula I: Provided herein is TIFF2025504682000001.tif32128, and pharma- ceutically acceptable salts thereof; During the ceremony: The subscript n is an integer ranging from 10 to 100; Each R 1 is independently C 1~6 is alkyl; Each R 2 is hydrogen, C 1~6 Alkyl, and C 1~6 acyl; (i) Z is -S(CH2)2C(O)-; R 3 -NR 3a R 3b , -NR 3a CH2CH(R 3b )2, -CH(R 3b )2, and -CH2CH(R 3b )2 selected from the group consisting of; or (ii) Z is -Z 1 -OC(O)-, -Z 1 -NHC(O)-, -Z 1 -S(O)2-, and -Z 1 -OCH2-, Z 1 is selected from the group consisting of a covalent bond, an oligo(ethylene glycol) diradical, and a poly(ethylene glycol) diradical; R 3 Ha-NR 3a R 3b and -CH(R 3b )2 selected from the group consisting of; In the formula, each R 3a and R 3b is hydrogen, C 6~22 Alkyl, C 6~22 Alkenyl, and C 6~22 alkynyl; R 3a and R 3b one or more non-adjacent CH groups in may be independently replaced with oxygen; However, at least one R 3aor R 3b But, C 6~22 Alkyl, C 6~22 Alkenyl, and C 6~22 alkynyl; R 3a or R 3b One or more non-adjacent CH2 groups in may be independently replaced with oxygen.

[0007] In some embodiments, R 1 is ethyl. In some embodiments, R 1 is methyl. In some embodiments, each R 1 is independently selected from the group consisting of methyl and ethyl.

[0008] In some embodiments, the subscript n is an integer ranging from about 15 to about 55. In some embodiments, the poly(alkyloxazoline) portion of the conjugate has a number average molecular weight ranging from about 2,000 Da to about 5,000 Da.

[0009] In some embodiments, the poly(alkyloxazoline)-lipid conjugate has a structure according to Formula IIa: TIFF2025504682000002.tif32128 or a pharma- ceutically acceptable salt thereof, During the ceremony: R 2 is selected from the group consisting of hydrogen and methyl; R 3a is hydrogen, C 12~18 Alkyl, and C 12~18 alkenyl; R 3b is C 12~18 Alkyl and C 12~18 alkenyl.

[0010] In certain aspects, the present disclosure provides lipid nanoparticles comprising a poly(alkyloxazoline)-lipid conjugate described herein (e.g., according to Formula I). ​​In some embodiments, the lipid nanoparticles further comprise a cationic lipid, a neutral lipid (e.g., a phospholipid), a sterol (e.g., cholesterol), or a combination thereof. In some embodiments, the lipid nanoparticles further comprise a nucleic acid. In certain embodiments, the nucleic acid comprises RNA (e.g., mRNA). In some embodiments, the nucleic acid is fully encapsulated in the lipid nanoparticle. In some embodiments, the lipid nanoparticles have an average diameter ranging from 40 nm to 150 nm.

[0011] In some aspects, the present disclosure provides a method for producing a method for manufacturing a semiconductor device comprising: (a) Nucleic acid; (b) cationic lipids constituting 30 mol %–80 mol % of the total lipids present in the lipid nanoparticles; (c) neutral lipids; (d) sterols; and (e) Poly(alkyloxazoline)-lipid conjugates that constitute 0.1 mol % to 10 mol % of the total lipids present in the lipid nanoparticles The present invention provides a lipid nanoparticle comprising:

[0012] In some embodiments, the cationic lipids comprise 40 mol% to 70 mol% of the total lipids present in the lipid nanoparticles. In some embodiments, the cationic lipids comprise 45 mol% to 65 mol% of the total lipids present in the lipid nanoparticles. In some embodiments, the cationic lipids comprise 45 mol% to 60 mol% of the total lipids present in the lipid nanoparticles. In some embodiments, the cationic lipids comprise 50 mol% to 60 mol% of the total lipids present in the lipid nanoparticles.

[0013] In some embodiments, the neutral lipid comprises 3 mol% to 20 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the neutral lipid comprises 5 mol% to 15 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the neutral lipid comprises 8 mol% to 12 mol% of the total lipid present in the lipid nanoparticle. In certain embodiments, the neutral lipid comprises a phospholipid.

[0014] In some embodiments, the sterol comprises 10 mol% to 60 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the sterol comprises 20 mol% to 50 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the sterol comprises 30 mol% to 40 mol% of the total lipid present in the lipid nanoparticle. In certain embodiments, the sterol comprises cholesterol.

[0015] In some embodiments, the poly(alkyloxazoline)-lipid conjugate comprises 0.1 mol% to 5 mol% of the total lipid present in the lipid nanoparticle. In some embodiments, the poly(alkyloxazoline)-lipid conjugate comprises 0.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticle. In certain embodiments, the poly(alkyloxazoline)-lipid conjugate comprises a poly(alkyloxazoline)-lipid conjugate described herein (e.g., according to Formula I).

[0016] In certain embodiments, the cationic lipids constitute 50 mol% to 60 mol% of the total lipids present in the lipid nanoparticles, the neutral lipids constitute 8 mol% to 12 mol% of the total lipids present in the lipid nanoparticles, the sterols constitute 30 mol% to 40 mol% of the total lipids present in the lipid nanoparticles, and the poly(alkyloxazoline)-lipid conjugates constitute 0.5 mol% to 3 mol% of the total lipids present in the lipid nanoparticles.

[0017] In some embodiments, the nucleic acid comprises RNA. In certain embodiments, the RNA comprises mRNA. In some embodiments, the nucleic acid is fully encapsulated in the lipid nanoparticle. In some embodiments, the lipid nanoparticle has an average diameter in the range of 40 nm to 150 nm.

[0018] In certain aspects, the present disclosure provides a pharmaceutical composition comprising the lipid nanoparticles described herein and a pharma- ceutical acceptable carrier.In some embodiments, the pharmaceutical composition is formulated for intravenous, intramuscular, pulmonary, intracerebral, intrathecal, or intranasal administration.

[0019] In some aspects, the present disclosure provides a method for introducing nucleic acid into a cell, comprising contacting the cell with lipid nanoparticles or pharmaceutical compositions described herein.In other aspects, the present disclosure provides a method for delivering nucleic acid to a subject, comprising administering lipid nanoparticles or pharmaceutical compositions described herein to the subject.In a further aspect, the present disclosure provides a method for preventing or treating disease or disorder in a subject in need thereof, comprising administering lipid nanoparticles or pharmaceutical compositions described herein to the subject.In some embodiments, the disease or disorder is a viral infection, a liver disease or disorder, a lung disease or disorder, a CNS disease or disorder, or cancer.

[0020] Other objects, features and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Detailed Description I. Introduction The present inventors have designed novel polymer-conjugated lipids comprising poly(alkyloxazoline) polymers, such as poly(2-ethyl 2-oxazoline) (PEOZ). The present inventors have conjugated the polymers to a variety of different lipid moieties, resulting in a series of poly(alkyloxazoline)-lipid conjugates. Such lipid conjugates have been formulated into lipid nanoparticles containing nucleic acids, such as mRNA payloads, and characterized, as described in the Examples herein.

[0022] The inventors have discovered that the lipid conjugates described herein form effective lipid nanoparticles with favorable physicochemical properties, such as desirable particle size and low polydispersity, as well as high encapsulation efficiency of nucleic acid (e.g., mRNA) payload. In vivo studies have demonstrated that the lipid nanoparticle formulations described herein mediate high levels of activity at the target site, exhibit similar or improved activity compared to PEG-lipid conjugates, and have lower non-specific (off-target) activity. The lipid nanoparticle formulations described herein contain similar or lower cytokine levels compared to PEG-lipid conjugates, thereby having a more favorable immunostimulatory profile. The lipid nanoparticle formulations described herein also exhibit improved plasma clearance rates compared to PEG-lipid conjugates, resulting in shorter residence time in plasma and mitigating antibody responses upon repeated dose administration. Further in vivo studies have demonstrated that the lipid nanoparticle formulations described herein can be successfully incorporated into vaccine platforms to deliver antigen-encoding mRNA and induce antigen-specific immunogenicity to a similar or greater extent compared to PEG-lipid conjugates. Thus, lipid nanoparticles comprising poly(alkyloxazoline)-lipid conjugates offer a more favorable activity and tolerability profile than particles comprising PEG-lipid conjugates.

[0023] II. Definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, reference to "a conjugate" optionally includes a combination of two or more such molecules, and so forth.

[0024] As used herein, the terms "about" and "approximately," when used to modify a quantity specified in a numerical value or range, indicate a numerical value and reasonable deviations from the value known to one of ordinary skill in the art, e.g., ±20%, ±10%, or ±5%, within the intended meaning of the recited value.

[0025] As used herein, the term "alkyl," by itself or as part of another substituent, refers to a straight or branched chain saturated aliphatic group having the number of carbon atoms indicated. Alkyl can have any number of carbons, e.g., C 1~2 , C 1~3 , C 1~4 , C 1~5 , C 1~6 , C 1~7 , C 1~8 , C 1~9 , C 1~10 , C 2~3 , C 2~4 , C 2~5 , C 2~6 , C 3~4 , C 3~5 , C 3~6 , C 4~5 , C 4~6 , and C 5~6 For example, C 1~6 Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. Alkyl can also refer to alkyl groups having up to 20 carbon atoms, such as, but not limited to, heptyl, octyl, nonyl, decyl, etc.

[0026] As used herein, the term "alkenyl," by itself or as part of another substituent, refers to an alkyl group having at least one carbon-carbon double bond.

[0027] As used herein, the term "alkynyl" by itself or as part of another substituent refers to an alkyl group having at least one carbon-carbon triple bond.

[0028] As used herein, the term "acyl," by itself or as part of another substituent, refers to the moiety -C(O)R, where R is an alkyl group.

[0029] As used herein, the terms "halo" and "halogen," by themselves or as part of another substituent, refer to a fluorine, chlorine, bromine, or iodine atom.

[0030] As used herein, the term "amino" refers to the moiety -NR2, where each R group is H or alkyl. The amino moiety can be ionized to form the corresponding ammonium cation.

[0031] As used herein, the term "sulfonyl" refers to the moiety -SO2R, where the R group is alkyl, haloalkyl, or aryl (e.g., phenyl, toluyl, naphthyl, etc.). The amino moiety can be ionized to form the corresponding ammonium cation. "Alkylsulfonyl" refers to an amino moiety where the R group is alkyl.

[0032] As used herein, the term "hydroxy" refers to the moiety --OH.

[0033] As used herein, the term "salt" refers to a compound that contains at least one cation (e.g., organic or inorganic cation) and at least one anion (e.g., organic or inorganic anion). Acid salts of lipids according to the present disclosure include, but are not limited to, mineral acid salts (e.g., salts formed using hydrochloric acid, hydrobromic acid, phosphoric acid, etc.), organic acid salts (e.g., salts formed using acetic acid, propionic acid, glutamic acid, citric acid, etc.), and quaternary ammonium salts (e.g., salts formed using methyl iodide, ethyl iodide, etc.). Acidic functional groups can be contacted with base to produce base salts, such as alkali and alkaline earth metal salts (e.g., sodium, lithium, potassium, calcium, and magnesium salts), and ammonium salts (e.g., ammonium, trimethyl-ammonium, diethylammonium, and tris-(hydroxymethyl)-methyl-ammonium salts).

[0034] In some embodiments, the neutral form of the compound can be regenerated, if desired, by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. In some embodiments, the parent form of the compound may differ from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salt forms may be identical to the parent form of the compound.

[0035] "Pharmaceutically acceptable" means that the excipient is compatible with other ingredients of the formulation and is not harmful to the recipient. For example, it is understood that pharma-ceutically acceptable excipients and salts are non-toxic. Useful pharmaceutical excipients include, but are not limited to, solvents, diluents, pH adjusters, and solubilizers.

[0036] The term "nucleic acid" refers to a polymer that contains at least two deoxyribonucleotides or ribonucleotides, either in single-stranded or double-stranded form, and includes DNA and RNA. DNA (e.g., ssDNA or dsDNA) may be in the form of, for example, antisense molecules, plasmid DNA, pre-enriched DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. The RNA (e.g., ssRNA or dsRNA) may be in the form of, for example, messenger RNA (mRNA), interfering RNA (e.g., small interfering RNA (siRNA), small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), guide RNA (gRNA), self-amplifying RNA, tRNA, rRNA, viral RNA (vRNA), and combinations thereof. In some embodiments, the nucleic acid is a plasmid from which an RNA such as an mRNA or interfering RNA is transcribed. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, natural, and non-natural, and which contain similar linkages as the reference nucleic acid. The analogs have the following characteristics: analogs that are easily modified by a single codon, such as a nucleotide sequence, ... et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).A "nucleotide" comprises the sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate group. A "base" includes purines and pyrimidines, which further include the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

[0037] As used herein, the term "lipid particle", "lipid nanoparticle", or "LNP" refers to a particle that comprises poly(alkyloxazoline)-lipid conjugate. The lipid particle may comprise additional lipid components, such as cationic lipids and one or more non-cationic lipids (e.g., phospholipids and / or sterols), and may further comprise nucleic acid, which may be encapsulated within the particle. In one embodiment, the nucleic acid is at least 50% encapsulated within the particle; in one embodiment, the nucleic acid is at least 75% encapsulated within the particle; in one embodiment, the nucleic acid is at least 90% encapsulated within the particle; in one embodiment, the nucleic acid is completely encapsulated within the particle. Lipid particles are very useful for systemic application, because they can show extended circulation life after intravenous injection, can accumulate at distant sites (e.g., sites physically separated from the administration site), and can mediate the expression of transfected genes or the silencing of target gene expression at these distant sites. The lipid particles typically have an average diameter of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 to about 80 nm, about 60 to about 80 nm, about 60 to about 90 nm, or about 70 to about 90 nm, and are substantially non-toxic. Furthermore, when present in the lipid particles, the nucleic acid is resistant to degradation by nucleases in aqueous solution. Lipid particles and methods for their preparation are disclosed, for example, in U.S. Patent Application Publication Nos. 2004 / 0142025 and 2007 / 0042031.

[0038] As used herein, the term "lipid-encapsulated" refers to lipid particles that provide nucleic acid (e.g., mRNA) completely encapsulated, partially encapsulated, or both. In one embodiment, the nucleic acid is completely encapsulated within the particle.

[0039] The term "cationic lipid" refers to a lipid species that carries a net positive charge at a selected pH, e.g., acidic pH or physiological pH. Non-limiting examples of cationic lipids are described in detail herein. In some cases, cationic lipids contain ionizable primary, secondary, or tertiary amine (e.g., pH-titratable) head groups. In one embodiment, cationic lipids promote encapsulation of negatively charged nucleic acid (e.g., mRNA) payloads during particle formation. In one embodiment, cationic lipids promote endosomal fusion and cytoplasmic release of payloads following cellular uptake of LNPs.

[0040] The term "non-cationic lipid" includes neutral lipids, sterols, and anionic lipids that are negatively charged at physiological pH, which exist either in uncharged form or in neutral zwitterionic form at selected pH (e.g., physiological pH).Non-limiting examples of non-cationic lipids are described in detail herein.Neutral lipids include, for example, phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine (LPE), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), and dilinoleoylphosphatidylcholine (DLPC). Sterols include, for example, cholesterol and its derivatives, such as cholestanol, cholestanone, cholestaneone, coprostanol, cholesteryl-2'-hydroxyethyl ether, and cholesteryl-4'-hydroxybutyl ether. Anionic lipids include, for example, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

[0041] As used herein, a "distant site" refers to a physically separated site, including sites that are not limited to adjacent capillary beds, but are widely distributed throughout a subject.

[0042] "Serum stability" in reference to lipid particles described herein means that the particles are not significantly degraded after exposure to serum or nuclease assays that would significantly degrade free DNA or RNA.Suitable assays include, for example, standard serum assays, DNAse assays, or RNAse assays.

[0043] As used herein, "systemic delivery" refers to the delivery of lipid particles that results in a wide biodistribution of nucleic acid, such as mRNA, in a subject. Systemic delivery means that a useful, preferably therapeutic amount of nucleic acid is exposed to most parts of the body. Obtaining a wide biodistribution generally requires a blood lifetime such that the nucleic acid is not rapidly degraded or eliminated (e.g., by first-pass organs (liver, lung, etc.) or by rapid non-specific cell binding) before reaching a target site distal to the administration site. Systemic delivery of lipid particles can be by any means known in the art, including, for example, intravenous, subcutaneous and intraperitoneal administration. In one embodiment, lipid particles are delivered intravenously.

[0044] As used herein, "local delivery" refers to the direct delivery of nucleic acid, such as mRNA, to target site in subject.For example, nucleic acid can be delivered locally by direct injection to disease site, such as tumor, or other target site, such as inflammation site, or target organ, such as liver, heart, pancreas, kidney, etc.

[0045] The terms "subject," "individual," and "patient," used interchangeably herein, refer to mammals, including but not limited to humans, non-human primates, rodents (e.g., rats, mice, and guinea pigs), rabbits, cows, pigs, horses, and other mammalian species. In one aspect, a subject, individual, or patient is a human.

[0046] III. Poly(alkyloxazoline)-lipid conjugates Poly(alkyloxazoline)-lipid conjugates according to formula I: Provided herein is TIFF2025504682000003.tif32128, and pharma- ceutically acceptable salts thereof; During the ceremony: The subscript n is an integer ranging from 10 to 100; Each R 1 is independently C 1~6 is alkyl; Each R 2 is hydrogen, C 1~6 Alkyl, and C 1~6 acyl; (i) Z is -S(CH2)2C(O)-; R 3 -NR 3a R 3b , -NR 3a CH2CH(R 3b )2, -CH(R 3b )2, and -CH2CH(R 3b )2 selected from the group consisting of; or (ii) Z is -Z 1 -OC(O)-, -Z 1 -NHC(O)-, -Z 1 -S(O)2-, and -Z 1 -OCH2-, Z 1 is selected from the group consisting of a covalent bond, an oligo(ethylene glycol) diradical, and a poly(ethylene glycol) diradical; R 3 -NR 3a R 3b and -CH(R 3b )2 selected from the group consisting of; In the formula, each R 3a and R 3b is hydrogen, C 6~22 Alkyl, C 6~22 Alkenyl, and C 6~22 alkynyl; R 3a and R 3b one or more non-adjacent CH groups in may be independently replaced with oxygen; However, at least one R 3aor R 3b But, C 6~22 Alkyl, C 6~22 Alkenyl, and C 6~22 alkynyl; R 3a or R 3b One or more non-adjacent CH2 groups in may be independently replaced with oxygen.

[0047] In some embodiments, R 1 is ethyl. In some embodiments, R 1 is methyl. In some embodiments, each R 1 is independently selected from the group consisting of methyl and ethyl.

[0048] In some embodiments, the subscript n is an integer ranging from about 15 to about 55. In some embodiments, the poly(alkyloxazoline) portion of the conjugate has a number average molecular weight ranging from about 2,000 Da to about 5,000 Da. Molecular weight can be determined by any suitable method, including, for example, by osmotic pressure, vapor pressure, light scattering, ultracentrifugation, or size exclusion chromatography. Size exclusion chromatography using an appropriately calibrated column can be used to determine the number average molecular weight M n is expressed as Equation 1: TIFF2025504682000004.tif8128 can be determined by Weight average molecular weight M w is expressed as Equation 2: TIFF2025504682000005.tif8128 can be determined by Where W is the total weight of the polymer, W i is the weight of the i-th polymer, and M i is the molecular weight of the i-th peak in the chromatogram, and N i is the molecular weight N i is the number of molecules having H i is the height of the i-th peak in the chromatogram.

[0049] In some embodiments, the poly(alkyloxazoline)-lipid conjugate has a structure according to Formula IIa: TIFF2025504682000006.tif32128 or a pharma- ceutically acceptable salt thereof; During the ceremony: R 2 is selected from the group consisting of hydrogen and methyl; R 3a is hydrogen, C 12~18 Alkyl, and C 12~18 alkenyl; R 3b is C 12~18 Alkyl and C 12~18 alkenyl.

[0050] In some embodiments, R 3a and R 3b is a poly(alkyloxazoline)-lipid conjugate according to formula IIa, 12~18 Alkyl and C 12~18 In some embodiments, R 3a and R 3b is independently C 12~18 In some embodiments, R of formula IIa is alkyl. 3a and R 3b is n-dodecyl (lauryl), n-tridecyl, n-tetradecyl (myristyl), n-pentadecyl, n-hexadecyl (cetyl), n-heptadecyl, or n-octadecyl (stearyl). 3a is H in formula IIa, R 3b is n-dodecyl (lauryl), n-tridecyl, n-tetradecyl (myristyl), n-pentadecyl, n-hexadecyl (cetyl), n-heptadecyl, or n-octadecyl (stearyl).

[0051] In some embodiments, the poly(alkyloxazoline)-lipid conjugate has a structure according to Formula IIb: TIFF2025504682000007.tif33147 or a pharma- ceutically acceptable salt thereof, During the ceremony: R 2 is selected from the group consisting of hydrogen and methyl; Each R 3c is C 12~18 Alkyl and C 12~18 In some embodiments, R in formula IIb is independently selected from the group consisting of aryl, aryl, and alkenyl. 3a and R 3b is n-dodecyl (lauryl), n-tridecyl, n-tetradecyl (myristyl), n-pentadecyl, n-hexadecyl (cetyl), n-heptadecyl, or n-octadecyl (stearyl).

[0052] The lipid conjugates can be synthesized using the methods described below, along with synthetic methods known in the art of organic synthetic chemistry or variations thereof. Preferred methods include, but are not limited to, those described in the Working Examples and the following schemes. Starting materials and reagents used in the preparation of the compounds of the present disclosure are available from commercial suppliers or can be prepared as described in Fieser and Fieser's Reagents for Organic Synthesis, Vol. 1-28 (Wiley, 2016); March's Advanced Organic Chemistry, 7 th Ed. (Wiley, 2013); and Larock's Comprehensive Organic Transformations, 2 nd Ed. (Wiley, 1999) or by methods known to those skilled in the art. The starting materials and intermediates of the reaction can be isolated and purified, if desired, using conventional techniques, including but not limited to filtration, chromatography, crystallization, distillation, and the like. Such materials can be characterized using conventional means, including measurement of physical constants and acquisition of spectral data.

[0053] Conjugates according to formula IIa and IIb can be formed by reacting N-hydroxysuccinimidyl ester terminated polymers according to formula III (eg, synthesized as described in WO2008 / 106186) with various amines. TIFF2025504682000008.tif33133

[0054] In some embodiments, carbamate-linked conjugates are provided, wherein Z is -Z 1 -OC(O)- and Z 1 is selected from the group consisting of a covalent bond and a poly(ethylene glycol) diradical; R 3 Ha-NR 3a R 3b The carbamate-linked conjugates can be prepared as shown in the following scheme: A hydroxy-terminated poly(alkyloxazoline) is first reacted with bis(4-nitrophenyl)carbonate to give a carbonate-terminated poly(alkyloxazoline), which can then be converted to a carbamate-linked product via reaction with an appropriate amine. The reaction is typically carried out in the presence of a base, such as potassium carbonate, sodium carbonate, sodium acetate, N,N-diisopropylethylamine, lutidine, including 2,6-lutidine, triethylamine, tributylamine, pyridine, lithium diisopropylamide, 2,6-di-tert-butylpyridine, 1,8-diazabicycloundec-7-ene (DBU), and the like. TIFF2025504682000009.tif55164

[0055] In some embodiments, urea-linked conjugates are provided, wherein Z is -Z 1 -NHC(O)-, and Z 1 is a covalent bond, and R 3 Ha-NR 3a R 3bThe urea-linked conjugates can be prepared as shown in the following scheme. First, the hydroxy-terminated poly(alkyloxazoline) is converted to an amine-terminated poly(alkyloxazoline) by reaction with phthalimide under Mitsunobu conditions, followed by reduction with hydrazine. The terminal amine is then converted to an amine by reaction with triphosgene and the amine HNR 3a R 3b and react with. TIFF2025504682000010.tif71163

[0056] In some embodiments, amide linked conjugates are provided, wherein Z is -Z 1 -NHC(O)-, and Z 1 is a covalent bond, and R 3 -CH(R 3b)2. As shown in the scheme below, the amine-terminated poly(alkyloxazoline) prepared as above can be acylated with a carboxylic acid to form an amide-linked conjugate. Coupling agents can be used to facilitate amide bond formation. Examples of suitable coupling agents include, but are not limited to, carbodiimides (e.g., N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), etc.), phosphonium salts (e.g., (benzotriazol-1-yloxy)-tripyrrolidinophosphonium hexafluorophosphate (PyBOP); bromotris(dimethylamino)-phosphonium hexafluorophosphate (BroP), etc.); guanidinium / uronium salts (e.g., methyl ethyl esters ... Salts (e.g., O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU); 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU); 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), etc.) and propanephosphonic anhydride. Coupling agents can be used with bases and / or catalysts, such as, but not limited to, pyridine and dimethylaminopyridine (DMAP). TIFF2025504682000011.tif65164

[0057] In some embodiments, ester-linked conjugates are provided, wherein Z is -Z 1 -OC(O)- and Z 1 is a covalent bond, and R 3 -CH(R 3b ) 2. Ester-linked conjugates can be prepared using coupling agents to link the carboxylic acid with the hydroxy-terminated poly(alkyloxazoline) as shown in the following scheme. TIFF2025504682000012.tif29164

[0058] In some embodiments, sulfonamide-linked conjugates are provided, wherein Z is -Z 1 -S(O)2-, and Z 1 is a covalent bond, and R 3 Ha-NR 3a R 3b As shown in the scheme below, hydroxyl-terminated poly(alkyloxazoline)s can be converted to sulfonyl chloride-terminated poly(alkyloxazoline)s using thionyl chloride and sodium sulfite. The sulfonyl chloride-terminated poly(alkyloxazoline)s can then be reacted with amines in the presence of base to provide sulfonamide-linked conjugates. TIFF2025504682000013.tif54164

[0059] In some embodiments, ether-linked conjugates are provided, wherein Z is -Z 1 -OCH2-, Z 1 is a covalent bond, and R 3 -CH(R 3b )2. Ether-linked conjugates can be prepared as shown below. Hydroxy-terminated poly(alkyloxazoline)s can be converted to sulfonate-terminated poly(alkyloxazoline), which is reacted with an alcohol to form the ether-linked conjugate. Sulfonates can be used, including but not limited to mesylate (methanesulfonic acid), triflate (trifluoromethanesulfonic acid), besylate (benzenesulfonate), tosylate (p-toluenesulfonate), and brosylate (4-bromobenzenesulfonate). TIFF2025504682000014.tif55164

[0060] In some embodiments, R 3a and each R 3b is a carbamate-linked conjugate, a urea-linked conjugate, an amide-linked conjugate, an ester-linked conjugate, a sulfonamide-linked conjugate, or an ether-linked conjugate,12~18 Alkyl and C 12~18 In some embodiments, R 3a and each R 3b is C 12~18 R is independently selected from alkyl. 3a and R 3b may be, for example, n-dodecyl (lauryl), n-tridecyl, n-tetradecyl (myristyl), n-pentadecyl, n-hexadecyl (cetyl), n-heptadecyl, or n-octadecyl (stearyl). 3a is H and R 3b is C 12~18 In some embodiments, two R 3b is C 12~18 alkyl.

[0061] In some embodiments, poly(alkyloxazoline)-poly(ethylene glycol) lipid conjugates are provided, wherein Z is -Z 1 -S(CH2)2C(O)-, -Z 1 -OC(O)-, -Z 1 -NHC(O)-, -Z 1 -S(O)2- or -Z 1 -OCH2-, Z 1 is an oligo(ethylene glycol) diradical or a poly(ethylene glycol) diradical. The oligo- or poly-(ethylene glycol) portion of the conjugate can contain from 2 to 200 ethylene glycol monomers. In some embodiments, the oligo- or poly-(ethylene glycol) Z 1 (e.g., Z 1The number-average molecular weight and / or weight-average molecular weight of the poly(ethylene glycol) may range from 0.025 to 5 kDa or 0.5 to 1 kDa. As shown below, poly(ethylene glycol) can be functionalized with terminal sulfonate groups, which can then be used to alkylate hydroxy-terminated poly(alkyloxazoline)s to form poly(alkyloxazoline)-poly(ethylene glycol) copolymers. The desired lipid conjugates can then be prepared from the copolymers using a variety of linking strategies, as described above. TIFF2025504682000015.tif58164

[0062] IV. Lipid particles In one aspect, the lipid particle comprises a poly(alkyloxazoline)-lipid conjugate according to formula I. In some embodiments, the lipid particle further comprises a cationic lipid, a neutral lipid, a sterol, or a combination thereof. In some embodiments, the lipid particle further comprises a nucleic acid. In another aspect, the lipid particle comprises a nucleic acid, a cationic lipid, a neutral lipid, a sterol, and a poly(alkyloxazoline)-lipid conjugate (e.g., according to formula I). ​​In some embodiments, the nucleic acid is completely encapsulated within the lipid portion of the lipid particle, such that the nucleic acid in the lipid particle is resistant to enzymatic degradation, for example, by nucleases or proteases, in aqueous solution. In some embodiments, the lipid particle is substantially non-toxic to mammals, such as humans.

[0063] In certain embodiments, the present disclosure provides lipid particle formulations comprising a plurality of lipid particles or a population of lipid particles. In some embodiments, the nucleic acid is fully encapsulated within the lipid portion of the lipid particle, such that the nucleic acid is present in about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100%, about 90% to about 100%, about 30% to about 95%, about 40% to about 95%, about 50% to about 95%, about 60% to about 95%, about 70% to about 95%, about 80% to about 95%, about 85% to about 95% of the lipid particles in the plurality of lipid particles or population of lipid particles. %, about 90% to about 95%, about 30% to about 90%, about 40% to about 90%, about 50% to about 90%, about 60% to about 90%, about 70% to about 90%, about 80% to about 90%, or at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any fraction or range therein) of the nucleic acid encapsulated therein.

[0064] In some embodiments, the lipid particles have an average diameter in the range of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 60 nm to about 100 nm, about 50 to about 80 nm, about 60 to about 80 nm, about 60 to about 90 nm, or about 70 to about 90 nm.

[0065] In some embodiments, the cationic lipid present in the lipid particle constitutes about 30 mol% to about 80 mol%, about 40 mol% to about 80 mol%, about 50 mol% to about 80 mol%, about 30 mol% to about 70 mol%, about 40 mol% to about 70 mol%, about 50 mol% to about 70 mol%, about 45 mol% to about 80 mol%, about 45 mol% to about 70 mol%, about 45 mol% to about 65 mol%, about 45 mol% to about 60 mol%, about 45 mol% to about 55 mol%, about 50 mol% to about 65 mol%, about 50 mol% to about 60 mol%, or about 55 mol% to about 65 mol% of the total lipid present in the particle. In some embodiments, the cationic lipid present in the lipid particle constitutes about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, or about 65 mol% of the total lipid present in the particle.

[0066] In some embodiments, the neutral lipids (e.g., phospholipids) present in the lipid particles comprise about 3 mol% to about 20 mol%, about 5 mol% to about 20 mol%, about 8 mol% to about 20 mol%, about 10 mol% to about 20 mol%, about 3 mol% to about 15 mol%, about 5 mol% to about 15 mol%, about 8 mol% to about 15 mol%, about 10 mol% to about 15 mol%, or about 8 mol% to about 12 mol% of the total lipids present in the particles. In some embodiments, the neutral lipids (e.g., phospholipids) present in the lipid particles comprise about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol% of the total lipids present in the particles.

[0067] In some embodiments, the sterol (e.g., cholesterol) present in the lipid particle constitutes about 10 mol% to about 60 mol%, about 20 mol% to about 60 mol%, about 30 mol% to about 60 mol%, about 40 mol% to about 60 mol%, about 20 mol% to about 50 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 50 mol%, about 40 mol% to about 50 mol%, about 20 mol% to about 45 mol%, about 25 mol% to about 45 mol%, about 30 mol% to about 45 mol%, about 35 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 40 mol%, or about 30 mol% to about 40 mol% of the total lipid present in the particle. In some embodiments, the sterol (e.g., cholesterol) present in the lipid particle constitutes about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, or about 50 mol% of the total lipid present in the particle.

[0068] In some embodiments, the poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) represents from about 0.1 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 1 mol% to about 10 mol%, about 1.5 mol% to about 10 mol%, about 2 mol% to about 10 mol%, about 2.5 mol% to about 10 mol%, about 3 mol% to about 10 mol%, about 4 mol% to about 10 mol%, about 5 mol% to about 10 mol%, about 0.1 mol% to about 5 ...0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 mol% to about 10 mol%, about 0.5 .3 mol% to about 5 mol%, about 0.5 mol% to about 5 mol%, about 1 mol% to about 5 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 5 mol%, about 2.5 mol% to about 5 mol%, about 3 mol% to about 5 mol%, about 0.1 mo 1% to about 3mol%, about 0.5mol% to about 3mol%, about 1mol% to about 3mol%, about 1.5mol% to about 3mol%, about 2mol% to about 3mol%, about 2.2mol% to about 3mol%, or about 2.5mol% to about 3mol%. In some embodiments, the poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) present in the lipid particle constitutes about 0.1 mol%, about 0.5 mol%, about 1 mol%, about 1.2 mol%, about 1.4 mol%, about 1.5 mol%, about 1.6 mol%, about 1.8 mol%, about 2 mol%, about 2.2 mol%, about 2.5 mol%, or about 3 mol% of the total lipid present in the particle.

[0069] In certain embodiments, the lipid particles comprise: cationic lipids that constitute from about 30 mol% to about 80 mol% of the total lipids present in the particle (e.g., from about 40 mol% to about 70 mol%, from about 45 mol% to about 65 mol%, from about 45 mol% to about 60 mol%, or from about 50 mol% to about 60 mol%) of the total lipids present in the particle; neutral lipids, such as phospholipids (e.g., from about 3 mol% to about 20 mol%, from about 5 mol% to about 15 mol%, or from about 8 mol% to about 12 mol%); sterols, such as cholesterol (e.g., from about 10 mol% to about 60 mol%, from about 20 mol% to about 50 mol%, or from about 30 mol% to about 40 mol%) of the total lipid present in the particle; and poly(alkyloxazoline)-lipid conjugates (e.g., according to Formula I) constituting from about 0.1 mol% to about 10 mol% of the total lipid present in the particle (e.g., from about 0.1 mol% to about 5 mol% or from about 0.5 mol% to about 3 mol% of the total lipid present in the particle).

[0070] In certain embodiments, the lipid particle comprises: a cationic lipid constituting about 50 mol% to about 60 mol% of the total lipid present in the particle; a neutral lipid (e.g., a phospholipid) constituting about 8 mol% to about 12 mol% of the total lipid present in the particle; a sterol (e.g., cholesterol) constituting about 30 mol% to about 40 mol% of the total lipid present in the particle; and a poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) constituting about 0.5 mol% to about 3 mol% of the total lipid present in the particle.

[0071] In one exemplary embodiment, the lipid particles comprise: cationic lipids, which comprise about 55 mol% (e.g., 54.6 mol%) of the total lipids present in the particles; neutral lipids (e.g., phospholipids), which comprise about 11 mol% (e.g., 10.9 mol%) of the total lipids present in the particles; sterols (e.g., cholesterol), which comprise about 33 mol% (e.g., 32.8 mol%) of the total lipids present in the particles; and poly(alkyloxazoline)-lipid conjugates (e.g., according to formula I), which constitute about 1.6 mol% of the total lipids present in the particles. In certain cases, the lipid particles are formulated into pharmaceutical compositions suitable for intravenous administration.

[0072] In another exemplary embodiment, the lipid particles comprise: a cationic lipid that constitutes about 50.0 mol% of the total lipid present in the particle; a neutral lipid (e.g., a phospholipid) that constitutes about 10 mol% of the total lipid present in the particle; a sterol (e.g., cholesterol) that constitutes about 38.5 mol% of the total lipid present in the particle; and a poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) that constitutes about 1.5 mol% of the total lipid present in the particle. In certain cases, the lipid particles are formulated into a pharmaceutical composition suitable for intramuscular administration.

[0073] In yet another exemplary embodiment, the lipid particle comprises: a cationic lipid constituting about 50.5 mol% of the total lipid present in the particle; a neutral lipid (e.g., a phospholipid) comprising about 10 mol% (e.g., 10.1 mol%) of the total lipid present in the particle; a sterol (e.g., cholesterol) comprising about 39 mol% (e.g., 38.9 mol%) of the total lipid present in the particle; and a poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) comprising about 0.5 mol% of the total lipid present in the particle.

[0074] In further exemplary embodiments, the lipid particle comprises: a cationic lipid comprising about 49 mol% (e.g., 49.2 mol%) of the total lipid present in the particle; a neutral lipid (e.g., a phospholipid) comprising about 10 mol% (e.g., 9.8 mol%) of the total lipid present in the particle; a sterol (e.g., cholesterol) comprising about 38 mol% (e.g., 37.9 mol%) of the total lipid present in the particle; and a poly(alkyloxazoline)-lipid conjugate (e.g., according to Formula I) constituting about 3.0 mol% of the total lipid present in the particle.

[0075] Any of a variety of cationic lipids can be used in the lipid particles described herein, either alone or in combination with one or more other cationic or non-cationic lipid species.

[0076] Cationic lipids useful in the lipid particles described herein can be any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxy ... lop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3.beta.-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleoylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLincarbDAP), DLinCDAP, 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-Dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy thio-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleoylamino)-1,2-propanediol (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and mixtures thereof.

[0077] In certain embodiments, the cationic lipid has the following structure: The compound is (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate with TIFF2025504682000016.tif16128.

[0078] In certain other embodiments, the cationic lipid has the following structure: I have TIFF2025504682000017.tif10128.

[0079] The non-cationic lipids used in the lipid particles described herein can be any of a variety of neutral uncharged zwitterionic or anionic lipids.

[0080] Neutral lipids include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine (LPE), phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine ( Exemplary classes of non-cationic lipids include phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups of these lipids are C 10 ~C 24 The acyl group may be derived from a fatty acid having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. In certain embodiments, the neutral lipid comprises DSPC.

[0081] Sterols are another exemplary class of non-cationic lipids, including cholesterol and its derivatives, such as cholestanol, cholestanone, cholestane, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the sterol comprises cholesterol.

[0082] Any poly(alkyloxazoline)-lipid conjugate according to formula I can be used in the lipid particles described herein. In an exemplary embodiment, the poly(alkyloxazoline) portion of the conjugate comprises a poly(2-ethyl 2-oxazoline) (PEOZ) polymer. In some embodiments, the poly(alkyloxazoline) portion of the conjugate has an average molecular weight ranging from about 2,000 Da to about 5,000 Da. In certain embodiments, the poly(alkyloxazoline) portion of the conjugate has an average molecular weight of about 5,000 Da. In some embodiments, the lipid portion of the conjugate comprises one or two independently selected C 12~18 In certain embodiments, the lipid portion of the conjugate comprises one or two independently selected C 12 , C 14 , C 16 , or C 18 Further poly(alkyloxazoline)-lipid conjugates suitable for use in the lipid particles described herein include the poly(alkyloxazoline)-dialkyloxypropyl conjugates disclosed in US Patent Application Publication No. 2011 / 0313017 and the poly(alkyloxazoline)-phospholipid conjugates disclosed in International Publication No. WO2010 / 006282.

[0083] V. Nucleic acids In certain embodiments, the lipid particles described herein are combined with nucleic acid, resulting in nucleic acid-lipid particles.In some embodiments, the nucleic acid is fully encapsulated in the lipid particles.As used herein, the term "nucleic acid" includes any oligonucleotide or polynucleotide, with fragments having up to 60 nucleotides generally referred to as oligonucleotides, and longer fragments referred to as polynucleotides.Nucleic acid can be administered alone in the lipid particles described herein, or in combination (e.g., co-administered) with lipid particles containing peptides, polypeptides, or small molecules, such as conventional drugs.

[0084] As used herein, the term "polynucleotide" and "oligonucleotide" refer to a polymer or oligomer of nucleotide or nucleoside monomers that are composed of natural bases, sugars, and intersugar (backbone) bonds.The term "polynucleotide" and "oligonucleotide" also include polymers or oligomers that contain non-natural monomers or portions thereof that function similarly.Such modified or substituted polynucleotides and oligonucleotides are often preferred over natural forms, for example, due to properties such as enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.

[0085] Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. Deoxyribooligonucleotides consist of a five-carbon sugar called deoxyribose covalently linked to phosphates at the 5' and 3' carbons of the sugar to form alternating, unbranched polymers. Ribooligonucleotides consist of a similar repeating structure in which the five-carbon sugar is ribose.

[0086] The nucleic acid present in the lipid particles described herein includes any known nucleic acid form.The nucleic acid used herein can be single-stranded DNA or RNA (e.g., ssDNA or ssRNA) or double-stranded DNA or RNA (e.g., dsDNA or dsRNA) or DNA-RNA hybrid.Single-stranded nucleic acids include, for example, mRNA, guide RNA (gRNA), antisense oligonucleotides, ribozymes, mature miRNA, and triplex-forming oligonucleotides.Examples of double-stranded DNA include, for example, structural genes, genes including control and termination regions, and self-replicating systems, for example, viral DNA or plasmid DNA.Examples of double-stranded RNA include, for example, siRNA and other RNAi agents, for example, aiRNA and pre-miRNA.

[0087] Nucleic acids can be of various lengths, generally depending on the particular form of the nucleic acid. For example, in certain embodiments, mRNA, plasmids, or genes can be from about 1,000 to about 100,000 nucleotides in length. In certain embodiments, oligonucleotides can range in length from about 10 to about 100 nucleotides. In various related embodiments, single-stranded, double-stranded, and triple-stranded oligonucleotides can range in length from about 10 to about 60 nucleotides, about 15 to about 60 nucleotides, about 20 to about 50 nucleotides, about 15 to about 30 nucleotides, or about 20 to about 30 nucleotides in length.

[0088] In certain embodiments, an oligonucleotide (or a strand thereof) specifically hybridizes to a target polynucleotide sequence or is complementary to a target polynucleotide sequence. As used herein, the terms "specifically hybridizable" and "complementary" refer to a sufficient degree of complementarity such that stable and specific binding occurs between a DNA or RNA target and an oligonucleotide. It will be understood that an oligonucleotide does not need to be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. In some embodiments, an oligonucleotide is specifically hybridizable when the binding of the oligonucleotide to the target sequence interferes with the normal function of the target sequence, causing its loss of utility or expression, and has a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under the conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatments, or under the conditions in which the assay is performed in the case of in vitro assays. Thus, an oligonucleotide can contain one, two, three or more base substitutions compared to the region of the gene or mRNA sequence to which it targets or to which it specifically hybridizes.

[0089] A. mRNA Certain embodiments provide compositions comprising the lipid particles described herein and methods of use thereof for expressing one or more mRNA molecules (e.g., a cocktail of mRNA molecules) in cells (e.g., cells in the human body). The mRNA molecules encode one or more polypeptides that are expressed in the cells. Lipid particle formulations comprising the mRNA molecules described herein are useful for a variety of applications, including protein replacement therapy, vaccines, cancer immunotherapy, and gene editing. In some embodiments, the lipid particles described herein are used to treat disease, and expression of the polypeptide encoded by the mRNA molecules in a diseased organism (e.g., a mammal, such as a human) ameliorates one or more symptoms of the disease. The compositions and methods described herein are particularly useful for treating human diseases caused by the absence or reduced levels of functional polypeptides in the human body. In other embodiments, the lipid particles described herein are used as vaccines to prevent disease, and expression of the polypeptide encoded by the mRNA molecules in an organism (e.g., a mammal, such as a human) induces immunity against the disease. The compositions and methods described herein are particularly useful for preventing infectious diseases caused by pathogens such as viruses (e.g., coronaviruses such as SARS-CoV-2) by expressing antigenic polypeptides (e.g., from mRNA molecules encoding viral proteins, such as S (spike), E (envelope), M (membrane), or N (nucleocapsid) proteins, or antigenic fragments thereof) to produce an immune response in an organism (e.g., a mammal, such as a human) by stimulating the adaptive immune system to generate antibodies that target the pathogen. In yet other embodiments, the lipid-loaded particles described herein are used as vaccines to treat disease, where expression of the polypeptides encoded by the mRNA molecules in an organism (e.g., a mammal, such as a human) induces an immune response against diseased cells.The compositions and methods described herein are particularly useful for treating cancer by expressing antigenic polypeptides (e.g., from mRNA molecules encoding tumor-specific antigens or antigenic fragments thereof) to stimulate an adaptive immune response to generate antibodies that target and destroy cancer cells.

[0090] In some embodiments, the mRNA molecules are completely encapsulated in lipid particles.For formulations that include mRNA cocktails, different types of mRNA species (e.g., mRNAs with different sequences) present in the cocktail can be co-encapsulated in the same particle, or each type of mRNA species present in the cocktail can be encapsulated in a separate particle.The mRNA cocktail can be formulated into particles as described herein using a mixture of two or more individual mRNAs (each with a unique sequence) at the same, similar, or different concentrations or molar ratios.In one embodiment, a cocktail of mRNAs (corresponding to multiple mRNAs with different sequences) is formulated using the same, similar, or different concentrations or molar ratios of each mRNA species, and the different types of mRNAs are co-encapsulated in the same particle. In another embodiment, each type of mRNA species present in the cocktail is encapsulated in a different particle at the same, similar, or different mRNA concentration or molar ratio, and the particles thus formed (each containing a different mRNA payload) are administered separately (e.g., at different times according to a prophylactic or therapeutic regimen) or are combined and administered together as a single unit dose (e.g., with a pharma- ceutically acceptable carrier).In certain embodiments, the lipid particles are serum stable, resistant to nuclease degradation, and substantially non-toxic to mammals, such as humans.

[0091] 1. Modification of mRNA The mRNA molecules present in the lipid particles can contain one, two, or more than two nucleoside modifications. In some embodiments, the modified mRNA exhibits reduced degradation in a cell into which the mRNA is introduced, compared to the corresponding unmodified mRNA.

[0092] In some embodiments, modified nucleosides include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl- Examples of pseudouridine include 1-methyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0093] In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl- These include pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0094] In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyl adenosine, N6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0095] In certain embodiments, the modified nucleoside is 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine, 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine. The α-thio-substituted phosphate moiety is provided to provide stability to RNA polymers through unnatural phosphorothioate backbone linkages. Phosphorothioate RNA has increased nuclease resistance in cellular environments, which in turn leads to longer half-lives. Phosphorothioate-linked nucleic acids are also expected to reduce innate immune responses through weaker binding / activation of cellular innate immune molecules.

[0096] In certain aspects, for example, when precise timing of protein production is desired, it is desirable to intracellularly degrade modified nucleic acids introduced into cells. Thus, the present disclosure provides modified nucleic acids that contain a degradation domain that can act in a directed manner within a cell.

[0097] In other embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

[0098] 2. Any mRNA component In further embodiments, the mRNA molecule present in the lipid particle can include other optional components. These optional components include, but are not limited to, untranslated regions, Kozak sequences, intron nucleotide sequences, internal ribosome entry sites (IRES), caps, and polyA tails. For example, it can include 5' untranslated regions (UTRs) and / or 3' UTRs, either or both of which can independently include one or more different nucleoside modifications. In such embodiments, nucleoside modifications can be present in translatable regions. Also provided are mRNA molecules that include Kozak sequences. Additionally provided herein are mRNA molecules that include one or more intron nucleotide sequences that can be excised from the mRNA sequence.

[0099] a. Untranslated Region (UTR) The untranslated region (UTR) of a gene is transcribed but not translated. 5'UTR starts from the transcription start point and continues to the start codon but does not include the start codon; whereas, 3'UTR starts immediately after the stop codon and continues to the transcription termination signal. There is increasing evidence of the regulatory role played by UTRs in terms of the stability and translation of mRNA molecules. To increase the stability of the molecule, the mRNA used in the lipid particles described herein can incorporate the regulatory function of UTR. Specific functions can also be incorporated to ensure the controlled downregulation of the transcript when the transcript is misdirected to an undesired tissue or organ site.

[0100] b. 5' cap formation The 5' cap structure of mRNA is involved in nuclear export, enhances mRNA stability, and binds to mRNA cap-binding protein (CBP), which is involved in mRNA stability and translational competence in cells through the binding of CBP with poly(A)-binding protein to form mature circular mRNA species. The cap further assists in the removal of 5'-proximal introns during mRNA splicing.

[0101] Endogenous mRNA molecules can be 5'-end capped to generate a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5' guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugar of the terminal and / or anti-terminal transcribed nucleotide at the 5' end of the mRNA can also be optionally 2'-O-methylated. 5' cap removal through hydrolysis and cleavage of the guanylate cap structure can target the mRNA molecule for degradation.

[0102] c. IRES sequence mRNAs containing an internal ribosome entry site (IRES) are also useful in the lipid particles described herein. An IRES can act as the only ribosome binding site or can act as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site can code for several peptides or polypeptides that are translated independently by ribosomes ("multicistronic mRNA"). When an mRNA is provided with an IRES, a second translatable region is optionally further provided. Examples of IRES sequences include, but are not limited to, those derived from picornaviruses (e.g., FMDV), plague viruses (e.g., CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), and cricket paralysis virus (CrPV).

[0103] d. PolyA tail During RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides, such as mRNA molecules, to increase stability. Immediately after transcription, the 3' end of the transcript can be cleaved to free a 3' hydroxyl. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. A process called polyadenylation adds a poly-A tail, which can be 100-250 residues long.

[0104] Generally, the length of the polyA tail is greater than 30 nucleotides in length. In some embodiments, the polyA tail is greater than 35 nucleotides in length (e.g., at least about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides, or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polyA tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the mRNA. In other embodiments, the polyA tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the total length of the mRNA.

[0105] 3. Generation of mRNA molecules Methods for isolating RNA, synthesizing RNA, hybridizing nucleic acids, making and screening cDNA libraries, and performing PCR are well known in the art (see, for example, Gubler and Hoffman, Gene, 25:263-269 (1983); Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989)), as are PCR methods (see, for example, U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)). Expression libraries are also well known to those skilled in the art. Additional basic texts that disclose the general methods of use in this disclosure include Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994).

[0106] 4. Application of mRNA Therapy The mRNA component of lipid particles described herein can be used to express polypeptide of interest.Certain human diseases are caused by the absence or dysfunction of a certain functional protein in the cell type in which the protein is normally present and active.Functional protein may be completely or partially absent, for example, due to the transcription of coding gene being inactive, or due to the existence of a mutation in coding gene that makes the protein completely or partially nonfunctional. Examples of human diseases caused by complete or partial inactivation of proteins include methylmalonic acid academia (caused by defective methylmalonyl-CoA mutase), glycogen storage disease type 1A (caused by defective catalytic subunit of glucose-6-phosphatase), glycogen storage disease type 1B (caused by lack of glucose-6-phosphate translocase), fragile X syndrome (caused by deficiency of the FMR1 protein), urea cycle disorders (caused by mutations in the ornithine transcarbamoylase (OTC) gene), Crigler-Najjar syndrome type 1 (caused by genetic mutations leading to lack of bilirubin uridine diphosphate glucuronosyltransferase (bilirubin-UGT)), alpha-1 antitrypsin deficiency (caused by mutations in the SERPINA1 gene) and alpha-1 antitrypsin deficiency (caused by mutations in the SERPINA1 gene). ), thrombotic thrombocytopenic purpura (caused by mutations in the disintegrin and metalloproteinase with thrombosponge type 1 motifs, member 13 (ADAMTS13) gene), acute intermittent porphyria (caused by genetic mutations at the PBGD locus), factor IX deficiency hemophilia B (caused by a deficiency of the factor IX (FIX) protein), X-linked severe combined immunodeficiency (X-SCID) (caused by one or more mutations in the gene encoding the common gamma chain protein, a component of the receptor for several interleukins involved in the development and maturation of B and T cells within the immune system), and X-linked adrenoleukodystrophy (X-ALD) (caused by one or more mutations in the peroxisomal membrane transporter protein gene called ABCD1).

[0107] In some embodiments, the mRNA component of the lipid particles described herein expresses an infectious disease antigen, such as a viral, bacterial, fungal, protozoan, and / or helminthic infectious disease antigen. Such vaccines comprising lipid particles with mRNA encoding the antigen are particularly useful for preventing or treating infectious diseases. In certain embodiments, the infectious disease antigen is a viral infectious disease antigen from coronavirus (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), influenza virus (e.g., influenza A, B and C viruses), filovirus (e.g., Ebola virus, Marburg virus), arenavirus (e.g., Lassa virus, Junin virus, Machupo virus, Gunarito virus, Sabia virus), Zika virus, rabies virus, rhinovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), hepatitis virus (e.g., hepatitis C virus), herpes simplex virus, human papillomavirus (HPV), or Epstein-Barr virus. In certain embodiments, the infectious disease antigen is a SARS-CoV-2 protein selected from the group consisting of S (spike) protein, E (envelope) protein, M (membrane) protein, N (nucleocapsid) protein, and antigenic fragments thereof. In certain cases, the generation of antigen-specific immunity from an mRNA vaccine requires the transfection of antigen-presenting cells, such as dendritic cells. Because dendritic cells are densely packed in skin tissue and skeletal muscle, administration is typically achieved by intradermal, intramuscular, or subcutaneous injection.

[0108] In some embodiments, the mRNA component of the lipid particles described herein expresses a tumor-associated antigen. In certain cases, following administration of the mRNA cancer vaccine to dendritic cells, cytotoxic T cells can target and destroy tumors. In other embodiments, the mRNA component of the lipid particles described herein expresses a chimeric antigen receptor (CAR) for CAR T cell therapy. Typically, a subject's T cells are isolated and transfected ex vivo with mRNA encoding a CAR, a protein fragment that is presented on the T cell surface and binds to a specific tumor epitope. Following reintroduction of the modified T cells into the subject, the CAR targets and kills tumor cells.

[0109] In some embodiments, the mRNA component of the lipid particle described herein expresses a gene editing nuclease. Examples of gene editing nucleases include CRISPR / Cas nucleases (e.g., Cas9, Cpf1), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and meganucleases. CRISPR-mediated gene editing requires a Cas nuclease responsible for DNA cleavage and a short guide RNA (gRNA) that directs the Cas nuclease to cleave DNA at a precise location. In some embodiments, the gRNA targets the Cas nuclease to a gene in the viral genome. As a non-limiting example, the viral genome is the SARS-CoV-2 genome, and the gene is selected from the group consisting of orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, and ORF10. In certain cases, the mRNA encoding a Cas nuclease, such as Cas9, and the gRNA are encapsulated in the same lipid particle. In other instances, the mRNA encoding a Cas nuclease and the gRNA are encapsulated in separate lipid particles.

[0110] B. siRNA The siRNA component of the lipid particles described herein can silence the expression of a target gene of interest. Each strand of the siRNA duplex is typically about 15 to about 60 nucleotides in length, preferably about 15 to about 30 nucleotides in length. In certain embodiments, the siRNA comprises at least one modified nucleotide. Modified siRNAs are generally less immunostimulatory than the corresponding unmodified siRNA sequence and retain RNAi activity against a target gene of interest. In some embodiments, the modified siRNA comprises at least one 2'OMe purine or pyrimidine nucleotide, such as a 2'OMe-guanosine, 2'OMe-uridine, 2'OMe-adenosine, and / or 2'OMe-cytosine nucleotide. The modified nucleotide can be present in one strand (i.e., sense or antisense) or both strands of the siRNA. The siRNA sequence may have overhangs (e.g., 3' or 5' overhangs as described in Elbashir et al., Genes Dev., 15:188 (2001) or Nykanen et al., Cell, 107:309 (2001)) or may lack overhangs (i.e., have blunt ends).

[0111] Suitable siRNA sequence can be identified by any means known in the art.Typically, the method described in Elbashir et al., Nature, 411:494-498 (2001) and Elbashir et al., EMBO J., 20:6877-6888 (2001) is combined with the rational design rule described in Reynolds et al., Nature Biotech., 22(3):326-330 (2004).

[0112] siRNA can be provided in several forms, for example, as one or more isolated short interfering RNA (siRNA) duplexes, as longer double-stranded RNA (dsRNA), or as siRNA or dsRNA transcribed from transcription cassette in DNA plasmid.siRNA can be chemically synthesized.Oligonucleotides including siRNA molecules can be synthesized using any of a variety of techniques known in the art, for example, those described in Usman et al., J. Am. Chem. Soc., 109:7845 (1987); Scaringe et al., Nucl. Acids Res., 18:5433 (1990); Wincott et al., Nucl. Acids Res., 23:2677-2684 (1995); and Wincott et al., Methods Mol. Bio., 74:59 (1997).

[0113] Examples of modified nucleotide include, but are not limited to, ribonucleotides with 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino or 2'-C-allyl group.Modified nucleotides with northern conformation, such as those described in Saenger, Principles of Nucleic Acid Structure, Springer-Verlag Ed. (1984), are also suitable for use in siRNA molecules. Such modified nucleotides include, but are not limited to, locked nucleic acid (LNA) nucleotides (e.g., 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'-O-(2-methoxyethyl) (MOE) nucleotides, 2'-methyl-thio-ethyl nucleotides, 2'-deoxy-2'-fluoro (2'F) nucleotides, 2'-deoxy-2'-chloro (2'Cl) nucleotides, and 2'-azido nucleotides. In certain cases, the siRNA molecules described herein comprise one or more G-clamp nucleotides. G-clamp nucleotides refer to modified cytosine analogs whose modification confers the ability to hydrogen bond to both the Watson-Crick and Hoogsteen faces of complementary guanine nucleotides in a duplex (see, for example, Lin et al., J. Am. Chem. Soc., 120:8531-8532 (1998)). Additionally, nucleotides having nucleotide base analogs, such as C-phenyl, C-naphthyl, other aromatic derivatives, inosine, azole carboxamide, and nitroazole derivatives, such as 3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole (see, e.g., Loakes, Nucl. Acids Res., 29:2437-2447 (2001)), can be incorporated into the siRNA molecules.

[0114] In certain embodiments, the siRNA molecule can further comprise one or more chemical modifications, such as terminal cap moieties, phosphate backbone modifications, etc. Examples of terminal cap moieties include, but are not limited to, inverted deoxyabasic residues, glyceryl modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 5'-5'-inverted nucleotide moieties, 5'- These include 5'-inverted abasic moieties, 3'-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphates, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidates, 5'-phosphoramidates, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioates, 5'-phosphorothioates, phosphorodithioates, and bridged or unbridged methylphosphonate or 5'-mercapto moieties (see, e.g., U.S. Pat. No. 5,998,203; Beaucage et al., Tetrahedron 49:1925 (1993)).Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and alkylsilyl substitutions (see, for example, H Hunziker et al., Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 331-417 (1995); Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Antisense Research, ACS, 24-39 (1994)). Such chemical modifications can occur at the 5'-end and / or 3'-end of the sense strand, antisense strand, or both strands of siRNA.

[0115] In some embodiments, the sense and / or antisense strands of the siRNA molecule can further comprise a 3'-end overhang having about 1 to about 4 (e.g., 1, 2, 3, or 4) 2'-deoxyribonucleotides and / or any combination of modified and unmodified nucleotides. Further examples of the types of modified nucleotides and chemical modifications that can be introduced into the siRNA molecule are described, for example, in British Patent No. GB2,397,818 B and US Patent Application Publication Nos. 20040192626, 20050282188, and 20070135372.

[0116] The siRNA molecule described herein can optionally comprise non-nucleotide in one or both strands of siRNA.As used herein, the term "non-nucleotide" refers to any group or compound, including sugar and / or phosphate substitution, that can be incorporated into nucleic acid strand instead of one or more nucleotide units, allowing the remaining base to show its activity.The group or compound does not comprise the commonly recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymine, and is therefore abasic in that it lacks the base at 1' position.

[0117] In other embodiments, chemical modification of siRNA comprises binding conjugate to siRNA molecule.For example, conjugate can be bound to 5' and / or 3' end of sense and / or antisense strand of siRNA via covalent bond such as biodegradable linker.For example, conjugate can be bound to siRNA via carbamate group or other linking group (see, for example, US Patent Application Publication Nos. 20050074771, 20050043219 and 20050158727).In certain cases, conjugate is a molecule that facilitates delivery of siRNA into cells. Examples of conjugate molecules suitable for attachment to siRNA include, but are not limited to, steroids, such as cholesterol, glycols, such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and derivatives thereof), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cellular receptors capable of mediating cellular uptake, and combinations thereof (see, e.g., U.S. Patent Application Publication Nos. 20030130186, 20040110296, and 20040249178; U.S. Patent No. 6,753,423). Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleavage agents, and cross-linking agent conjugate molecules described in U.S. Patent Application Publication Nos. 20050119470 and 20050107325. Still other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationically modified pyrimidines, cationic peptides, guanidinium groups, amidinium groups, cationic amino acid conjugate molecules described in U.S. Patent Application Publication No. 20050153337.Further examples include the conjugate molecules of hydrophobic groups, membrane active compounds, cell permeable compounds, cell targeting signals, interaction modifiers, and steric stabilizers described in US Patent Publication No. 20040167090. Further examples include the conjugate molecules described in US Patent Publication No. 20050239739. The type of conjugate used and the degree of conjugation to siRNA molecules can be evaluated for improving the pharmacokinetic profile, bioavailability, and / or stability of siRNA while retaining RNAi activity. Thus, those skilled in the art can use any of a variety of well-known in vitro cell culture or in vivo animal models to screen siRNA molecules with various conjugates to identify those with improved properties and full RNAi activity.

[0118] The siRNA component of lipid particles described herein can be used to downregulate or silence the translation (i.e., expression) of gene of interest.The gene of interest includes, but is not limited to, the gene related to virus infection and survival, the gene related to metabolic disease and disorder (e.g., liver disease and disorder), the gene related to tumorigenesis and cell transformation (e.g., cancer), the gene related to angiogenesis promotion, the immune regulator gene, for example, the one related to inflammatory and autoimmune response, the ligand receptor gene, and the gene related to neurodegenerative disorder.

[0119] Genes related to viral infection and survival include those expressed by viruses to bind to, enter, and replicate in cells. Viral sequences of particular interest include sequences from coronaviruses (e.g., SARS-CoV-1, SARS-CoV-2, MERS-CoV), influenza viruses (e.g., influenza A, B, and C viruses), filoviruses (e.g., Ebola virus, Marburg virus), arenaviruses (e.g., Lassa virus, Junin virus, Machupo virus, Gunarito virus, Sabia virus), Zika virus, rabies virus, rhinovirus, simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), hepatitis viruses (e.g., hepatitis C virus), herpes simplex virus, human papillomavirus (HPV), or Epstein-Barr virus. Exemplary coronavirus (e.g., SARS-CoV-2) nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding S (spike) protein, E (envelope) protein, M (membrane) protein, and N (nucleocapsid) protein. Exemplary filovirus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding structural proteins (e.g., VP30, VP35, nucleoprotein (NP), polymerase protein (L-pol)) and membrane-associated proteins (e.g., VP40, glycoprotein (GP), VP24). Exemplary influenza virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences encoding nucleoprotein (NP), matrix protein (M1 and M2), nonstructural proteins (NS1 and NS2), RNA polymerase (PA, PB1, PB2), neuraminidase (NA), and hemagglutinin (HA).Exemplary Hepatitis virus nucleic acid sequences that can be silenced include, but are not limited to, nucleic acid sequences involved in transcription and translation (e.g., En1, En2, X, P), and nucleic acid sequences encoding structural proteins (e.g., core proteins, e.g., C and C-related proteins, capsid, and envelope proteins, e.g., S, M, and / or L proteins, or fragments thereof).

[0120] Genes associated with metabolic diseases and disorders (e.g., disorders that target the liver and liver diseases and disorders) include, for example, dyslipidemia (e.g., liver X receptors, e.g., LXRα and LXRβ, farnesoid X receptor (FXR), sterol regulatory element binding protein (SREBP), site 1 protease (S1P), 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMG coenzyme-A reductase), apolipoprotein B (ApoB), apolipoprotein CIII (ApoC3), and apolipoprotein E (ApoC4). E); and diabetes (e.g., glucose 6-phosphatase). Those skilled in the art will recognize that genes related to metabolic diseases and disorders (e.g., liver-targeted diseases and disorders and liver diseases and disorders) include genes expressed in the liver itself and genes expressed in other organs and tissues. Silencing of sequences encoding genes related to metabolic diseases and disorders can be advantageously used in combination with the administration of conventional agents used to treat diseases or disorders.

[0121] Examples of gene sequences associated with tumor development and cell transformation (e.g., cancer or other neoplasms) include mitotic kinesins, such as Eg5 (KSP, KIF11); serine / threonine kinases, such as polo-like kinase 1 (PLK-1); tyrosine kinases, such as WEE1; inhibitors of apoptosis, such as XIAP; COP9 signalosome subunits, such as CSN1, CSN2, CSN3, CSN4, CSN5; CSN6, CSN7A, CSN7B, and CSN8; ubiquitin ligases, such as COP1 (RFWD2); and histone deacetylases, such as HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6, HDAC7, HDAC8, HDAC9, and the like.

[0122] Further examples of gene sequences associated with tumor development and cell transformation include translocation sequences, e.g., MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1, TLS-FUS, PAX3-FKHR, BCL-2, AML1-ETO, and AML1-MTG8; overexpressed sequences, e.g., multidrug resistance genes, cyclins, beta-catenin, telomerase genes, c-MYC, N-MYC, BCL-2, growth factor receptors (e.g., EGFR / ErbB1, ErbB2 / HER-2, ErbB3, and ErbB4); and mutated sequences, e.g., RAS.

[0123] Silencing of the sequence that codes for DNA repair enzymes is used in combination with the administration of chemotherapy.The gene that codes for the protein related to tumor migration is also an interesting target sequence, for example, integrin, selectin, and metalloproteinase.The above examples are not exclusive.Those skilled in the art will understand that any whole or partial gene sequence that facilitates or promotes tumor development or cell transformation, tumor growth, or tumor migration can be included as template sequence.

[0124] Pro-angiogenic genes can promote the formation of new blood vessels. Of particular interest is vascular endothelial growth factor (VEGF) or VEGFR. Anti-angiogenic genes can inhibit angiogenesis. These genes are particularly useful for treating cancers in which angiogenesis plays a role in the pathogenesis of the disease. Examples of anti-angiogenic genes include, but are not limited to, endostatin, angiostatin, and VEGFR2.

[0125] Immunomodulatory factor genes are genes that modulate one or more immune responses. Examples of immunomodulatory factor genes include, but are not limited to, cytokines, such as growth factors (e.g., TGF-α, TGF-β, EGF, FGF, IGF, NGF, PDGF, CGF, GM-CSF, SCF, etc.), interleukins (e.g., IL-2, IL-4, IL-12, IL-15, IL-18, IL-20, etc.), interferons (e.g., IFN-α, IFN-β, IFN-γ, etc.), and TNF. Fas and Fas ligand genes are also interesting immunomodulatory factor target sequences. Also included are genes that code for secondary signaling molecules in hematopoietic and lymphoid cells, such as Tec family kinases, such as Bruton's tyrosine kinase (Btk).

[0126] Cell receptor ligands include ligands that can bind to cell surface receptors (e.g., insulin receptors, EPO receptors, G protein-coupled receptors, receptors with tyrosine kinase activity, cytokine receptors, growth factor receptors, etc.) and modulate (e.g., inhibit, activate, etc.) physiological pathways in which the receptor is involved (e.g., modulation of glucose levels, blood cell development, mitogenesis, etc.). Examples of cell receptor ligands include, but are not limited to, cytokines, growth factors, interleukins, interferons, erythropoietin (EPO), insulin, glucagon, ligands of G protein-coupled receptors, etc. Templates that code for expansions of trinucleotide repeats (e.g., CAG repeats) are used to silence pathogenic sequences in neurodegenerative disorders caused by expansions of trinucleotide repeats, such as spinal and bulbar muscular atrophy and Huntington's disease.

[0127] Certain other target genes that can be targeted by nucleic acids (e.g., siRNA) to downregulate or silence expression of the genes include, but are not limited to, Actin, Alpha 2, Smooth Muscle, Aorta (ACTA2), alcohol dehydrogenase 1A (ADH1A), alcohol dehydrogenase 4 (ADH4), alcohol dehydrogenase 6 (ADH6), afamin (AFM), angiotensinogen (AGT), serine-pyruvate aminotransferase (AGXT), alpha-2-HS-glycoprotein (AHSG), aldo-ketoreductase family 1 member C4 (AKR1C4), serum albumin (ALB), alpha-1-microglobulin / bikunin precursor (AMBP), angiopoietin-related protein 3 (ANGP), and alpha-1-microglobulin / bikunin precursor (AMBP). TL3), serum amyloid P component (APCS), apolipoprotein A-II (APOA2), apolipoprotein B-100 (APOB), apolipoprotein C3 (APOC3), apolipoprotein C-IV (APOC4), apolipoprotein F (APOF), beta-2-glycoprotein 1 (APOH), aquaporin-9 (AQP9), bile acid-CoA: amino acid N-acyltransferase (BAAT), C4b-binding protein beta chain (C4BPB), putative function unknown protein encoded by LINC01554 (Putative uncharacterized protein) (C5orf27), complement factor 3 (C3), complement factor 5 (C5), complement component C6 (C6), complement component C8 alpha chain (C8A), complement component C8 beta chain (C8B), complement component C8 gamma chain (C8G), complement component C9 (C9), calmodulin-binding transcription activator 1 (CAMTA1), CD38 (CD38), complement factor B (CFB), complement factor H-related protein 1 (CFHR1), complement factor H-related protein 2 (CFHR2), complement factor H-related protein 3 (CFHR3), cannabinoid receptor 1 (CNR1), ceruloplasmin (CP), carboxypeptidase B2 (CPB2), connective tissue growth factor (CTGF), C-X-C motif chemokine 2 (CXCL2), cytochrome P450 1 A2 (CYP1A2), cytochrome P4502A6 (CYP2A6), cytochrome P450 2C8 (CYP2C8), cytochrome P450 2C9 (CYP2C9), cytochrome P450 family 2 subfamily D member 6 (CYP2D6), cytochrome P450 2E1 (CYP2E1), phylloquinone omega-hydroxylase CYP4F2 (CYP4F2), 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase (CYP8B1), dipeptidyl peptidase 4 (DPP4), coagulation factor 12 (F12), coagulation factor II (thrombin) (F2), coagulation factor LX (F9), fibrinogen alpha chain (FGA), fibrinogen beta chain (FGB), fibrinogen gamma chain ( FGG), fibrinogen-like 1 (FGL1), flavin-containing monooxygenase 3 (FM03), flavin-containing monooxygenase 5 (FM05), group-specific component (vitamin D binding protein) (GC), growth hormone receptor (GHR), glycine N-methyltransferase (GNMT), hyaluronan-binding protein 2 (HABP2), hepcidin antimicrobial peptide (HAMP), hydroxyacid oxidase (glycolate oxidase) 1 (HAO1), HG F-activating factor (HGFAC), haptoglobin-related protein; haptoglobin (HPR), hemopexin (HPX), histidine-rich glycoprotein (HRG), hydroxysteroid (11-beta) dehydrogenase 1 (HSD11B1), hydroxysteroid (17-beta) dehydrogenase 13 (HSD17B13), inter-alpha trypsin inhibitor heavy chain H1 (ITIH1), inter-alpha trypsin inhibitor heavy chain H2 (ITIH2), Inter-alpha trypsin inhibitor heavy chain H3 (ITIH3), inter-alpha trypsin inhibitor heavy chain H4 (ITIH4), prekallikrein (KLKB1), lactate dehydrogenase A (LDHA), liver-expressed antimicrobial peptide 2 (LEAP2), leukocyte-derived chemotaxin 2 (LECT2), lipoprotein(a) (LPA), mannan-binding lectin serine peptidase 2 (MASP2), S-adenosylmethionine synthase isoform type 1 (MAT1A), NADPH oxidase 4 (NOX4), poly[ADP-ribose] polymerase 1 (PARP1), paraoxonase 1 (PON1), paraoxonase 3 (PON3), vitamin K-dependent protein C (PROC), retinol dehydrogenase 16 (RDH16), serum amyloid A4, constitutive (SAA4), serine dehydratase (SDS), serpin family A member 1 (SERPINA1), serpin A11 (SERPINA11), kallistatin (SERPINA4), corticosterone-binding protein 1 (CRP1), and phosphodiesterase 2 (GMP). Corsteroid-binding globulin (SERPINA6), antithrombin-PI (SERPINC1), heparin cofactor 2 (SERPIND1), serpin family H member 1 (SERPINH1), solute carrier family 5 member 2 (SLC5A2), sodium / bile acid cotransporter (SLC10A1), solute carrier family 13 member 5 (SLC13A5), solute carrier family 22 member 1 (SLC22A1), solute carrier family 25 member 47 (SLC25 A47), solute carrier family 2, facilitative glucose transporter member 2 (SLC2A2), sodium-linked neutral amino acid transporter 4 (SLC38A4), solute carrier organic anion transporter family member 1B1 (SLCO1B1), sphingomyelin phosphodiesterase 1 (SMPD1), bile salt sulfotransferase (SEILT2A1), tyrosine aminotransferase (TAT), tryptophan 2,3-dioxygenase (TDO2), UDP-glucuronosyltransferase 2 family, polypeptide B10 (UGT2B10), UDP-glucuronosyltransferase 2 family, polypeptide B15 (UGT2B15), UDP-glucuronosyltransferase 2 family, polypeptide B4 (UGT2B4), and vitronectin (VTN).

[0128] C. aiRNA Similar to siRNA, asymmetric interfering RNA (aiRNA) can lead to effective silencing of various genes in mammalian cells by recruiting RNA-induced silencing complex (RISC) and mediating sequence-specific cleavage of the target sequence between nucleotides 10 and 11 relative to the 5' end of the antisense strand (Sun et al., Nat. Biotech., 26:1379-1382 (2008)). Typically, aiRNA molecules contain a short RNA duplex with a sense strand and an antisense strand, and the duplex contains overhangs at the 3' and 5' ends of the antisense strand. aiRNAs are generally asymmetric because the sense strand is short at both ends when compared to the complementary antisense strand. In some aspects, aiRNA molecules can be designed, synthesized, and annealed under conditions similar to those used for siRNA molecules. As a non-limiting example, aiRNA sequences can be selected and generated using the methods described above for selecting siRNA sequences.

[0129] In another embodiment, aiRNA duplexes of various lengths (e.g., about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 base pairs, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 base pairs) can be designed with overhangs at the 3' and 5' ends of the antisense strand to target an mRNA of interest. In certain cases, the sense strand of the aiRNA molecule is about 10-25, 12-20, 12-19, 12-18, 13-17, or 14-17 nucleotides in length, more typically 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In certain other instances, the antisense strand of the aiRNA molecule is about 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length.

[0130] In some embodiments, the 5' antisense overhang comprises one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In other embodiments, the 3' antisense overhang comprises one, two, three, four, or more non-targeting nucleotides (e.g., "AA", "UU", "dTdT", etc.). In certain aspects, the aiRNA molecules described herein can comprise one or more modified nucleotides, for example, in the double-stranded (duplex) region and / or the antisense overhang. As non-limiting examples, the aiRNA sequence can comprise one or more of the modified nucleotides described above for the siRNA sequence.

[0131] In certain embodiments, the aiRNA molecule can include an antisense strand that corresponds to the antisense strand of an siRNA molecule, e.g., one of the siRNA molecules described herein. In other embodiments, the aiRNA molecule can be used to silence the expression of any of the target genes described above in the context of siRNA molecules, such as genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation, pro-angiogenic genes, immune regulator genes, e.g., those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. As a non-limiting example, antisense oligonucleotides can be used to silence the expression of SARS-CoV-2 genes encoding the S (spike) protein, E (envelope) protein, M (membrane) protein, or N (nucleocapsid) protein.

[0132] D. miRNA In general, microRNAs (miRNAs) are single-stranded RNA molecules, approximately 21-23 nucleotides in length, that regulate gene expression. Although miRNAs are encoded by genes from the DNA from which they are transcribed, miRNAs are not translated into proteins (non-coding RNA); instead, each primary transcript (pri-miRNA) is processed into a short stem-loop structure called pre-miRNA, and finally into a functional mature miRNA. Mature miRNA molecules are either partially or fully complementary to one or more messenger RNA (mRNA) molecules, and their main function is to downregulate gene expression.

[0133] Genes encoding miRNAs are much longer than the mature processed miRNA molecules. miRNAs are first transcribed in the nucleus of cells as primary transcripts or pri-miRNAs with caps and polyA tails, and are processed into short, approximately 70 nucleotide stem-loop structures known as pre-miRNAs. In animals, this processing is carried out by a protein complex known as the microprocessor complex, which consists of the nuclease Drosha and the double-stranded RNA-binding protein Pasha (Denli et al., Nature, 432:231-235 (2004)). These pre-miRNAs are then processed into mature miRNAs in the cytoplasm by interaction with the endonuclease Dicer, which also initiates the formation of the RNA-induced silencing complex (RISC) (Bernstein et al., Nature, 409:363-366 (2001). Either the sense or antisense strand of DNA can serve as a template to generate miRNAs.

[0134] When Dicer cleaves the stem loop of the pre-miRNA, two complementary short RNA molecules are formed, but only one is incorporated into the RISC complex. This strand is known as the guide strand and is selected by the catalytically active RNase Argonaute protein in the RISC complex based on the stability of its 5' end (Preall et al., Curr. Biol., 16:530-535 (2006)). The remaining strand, known as the anti-guide strand or passenger strand, is degraded as a RISC complex substrate (Gregory et al., Cell, 123:631-640 (2005)). After incorporation into the active RISC complex, the miRNA base pairs with its complementary mRNA molecule and induces the degradation and / or translational silencing of the target mRNA.

[0135] Mammalian miRNA molecules are usually complementary to a site in the 3'UTR of a target mRNA sequence. In certain instances, annealing of the miRNA to the target mRNA inhibits protein translation by blocking the protein translation machinery. In certain other instances, annealing of the miRNA to the target mRNA facilitates cleavage and degradation of the target mRNA through a process similar to RNA interference (RNAi). miRNAs can also target methylation of genomic sites corresponding to the target mRNA. In general, miRNAs function in association with a complement of proteins collectively referred to as miRNPs.

[0136] In certain aspects, the miRNA molecules described herein are about 15-100, 15-90, 15-80, 15-75, 15-70, 15-60, 15-50, or 15-40 nucleotides in length, more typically about 15-30, 15-25, or 19-25 nucleotides in length, and preferably about 20-24, 21-22, or 21-23 nucleotides in length. In certain other aspects, the miRNA molecule can include one or more modified nucleotides. As a non-limiting example, the miRNA sequence can include one or more of the modified nucleotides described above for the siRNA sequence.

[0137] In some embodiments, miRNA molecules can be used to silence the expression of any of the target genes described above in the context of siRNA molecules, such as genes associated with viral infection and survival, genes associated with metabolic diseases and disorders, genes associated with tumorigenesis and cell transformation, pro-angiogenic genes, immune regulator genes, such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. As a non-limiting example, miRNAs can be used to silence the expression of SARS-CoV-2 genes encoding the S (spike) protein, E (envelope) protein, M (membrane) protein, or N (nucleocapsid) protein.

[0138] In other embodiments, one or more agents that block the activity of miRNA that targets mRNA of interest are administered using lipid particles as described herein.Examples of blocking agents include but are not limited to steric blocking oligonucleotides, locked nucleic acid oligonucleotides, and morpholino oligonucleotides.Such blocking agents can directly bind to miRNA or bind to the miRNA binding site on target mRNA.

[0139] E. Antisense Oligonucleotides In some embodiments, the nucleic acid component of the lipid particles described herein is an antisense oligonucleotide to a target gene or sequence of interest. The term "antisense oligonucleotide" or "antisense" includes an oligonucleotide that is complementary to a target polynucleotide sequence. An antisense oligonucleotide is a single strand of DNA or RNA that is complementary to a selected sequence. An antisense RNA oligonucleotide prevents translation of the complementary RNA strand by binding to the RNA. An antisense DNA oligonucleotide can be used to target a specific complementary (coding or non-coding) RNA. Once binding occurs, the DNA / RNA hybrid can be degraded by the RNase H enzyme. In certain embodiments, the antisense oligonucleotide contains about 10 to about 60 nucleotides or about 15 to about 30 nucleotides. The term also encompasses antisense oligonucleotides that may not be exactly complementary to the desired target gene. Thus, the lipid particles described herein can be utilized when non-target specific activity is observed with the antisense or when an antisense sequence that contains one or more mismatches with the target sequence is most preferred for a particular application. The antisense oligonucleotides can comprise natural nucleotides, as well as non-natural or modified nucleotides (eg, modified nucleobases, modified internucleoside linkages, and / or modified sugars, such as those described herein).

[0140] Methods for generating antisense oligonucleotides are known in the art and can be readily adapted to generate antisense oligonucleotides targeting any polynucleotide sequence. Selection of an antisense oligonucleotide sequence specific for a given target sequence can be determined by analysis of the selected target sequence, as well as secondary structure, T mAntisense oligonucleotides can be selected based on their relative inability to form dimers, hairpins, or other secondary structures that reduce or prevent specific binding to target mRNA in host cells. Highly preferred target regions of mRNA include the region of or near the AUG translation initiation codon and sequences that are substantially complementary to the 5' region of mRNA. These secondary structure analyses and target site selection studies can be performed, for example, using OLIGO primer analysis software v.4 (Molecular Biology Insights) and / or BLASTN 2.0.5 algorithm software (Altschul et al., Nucleic Acids Res., 25:3389-402 (1997)).

[0141] In some embodiments, the antisense oligonucleotide component of the lipid particles described herein can be used to inhibit the expression or replication of a gene of interest. Genes of interest are described above in the context of siRNA molecules, and include, but are not limited to, genes associated with viral infection and survival, genes associated with metabolic diseases and disorders (e.g., liver diseases and disorders), genes associated with tumorigenesis and cell transformation (e.g., cancer), pro-angiogenic genes, immune regulator genes, such as those associated with inflammatory and autoimmune responses, ligand receptor genes, and genes associated with neurodegenerative disorders. As a non-limiting example, an antisense oligonucleotide can hybridize to a SARS-CoV-2 gene (e.g., orf1ab, S gene, ORF3a, E gene, M gene, ORF6, ORF7a, ORF8, N gene, or ORF10) and inhibit the expression or replication of the gene.

[0142] F. Ribozymes In some embodiments, the nucleic acid component of the lipid particles described herein is a ribozyme.Ribozymes are RNA-protein complexes with specific catalytic domains that have endonuclease activity.For example, many ribozymes accelerate phosphoester transfer reactions with high specificity, often cleaving only one of several phosphates in oligonucleotide substrates.This specificity is due to the need for substrates to bind to the internal guide sequence ("IGS") of ribozymes through specific base-pairing interactions prior to chemical reaction.

[0143] At least six basic types of naturally occurring enzymatic RNA molecules are currently known. Each is capable of catalyzing the hydrolysis of RNA phosphodiester bonds in trans (and thereby cleaving other RNA molecules) under physiological conditions. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of the enzymatic nucleic acid, which is held in close proximity to the enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes the target RNA through complementary base pairing, then binds to the target RNA, and once bound to the correct site, acts enzymatically to cleave the target RNA. Such strategic cleavage of the target RNA is believed to destroy its ability to direct the synthesis of an encoded protein. After the enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.

[0144] The enzymatic nucleic acid molecule can be formed, for example, with hammerhead, hairpin, hepatitis delta virus, group I intron or RNaseP RNA (combined with an RNA guide sequence), or Neurospora VS RNA motifs. Specific examples of hammerhead motifs are described, for example, in Rossi et al., Nucleic Acids Res., 20:4559-65 (1992). Examples of hairpin motifs are described, for example, in EP0360257, Hampel et al., Biochemistry, 28:4929-33 (1989); Hampel et al., Nucleic Acids Res., 18:299-304 (1990); and U.S. Patent No. 5,631,359. Examples of hepatitis delta virus motifs are described, for example, in Perrotta et al., Biochemistry, 31:11843-52 (1992). Examples of RNaseP motifs are described, for example, in Guerrier-Takada et al., Cell, 35:849-57 (1983). Examples of Neurospora VS RNA ribozyme motifs are described, for example, in Saville et al., Cell, 61:685-96 (1990); Saville et al., Proc. Natl. Acad. Sci. USA, 88:8826-30 (1991); Collins et al., Biochemistry, 32:2795-9 (1993). Examples of group I introns are described, for example, in U.S. Pat. No. 4,987,071. The important characteristics of an enzymatic nucleic acid molecule are that it has a specific substrate binding site that is complementary to one or more of the DNA or RNA regions of the target gene, and that it has a nucleotide sequence within or surrounding the substrate binding site that confers RNA cleavage activity to the molecule. Thus, ribozyme constructs need not be limited to the specific motifs mentioned herein.

[0145] Methods for generating ribozymes that target any polynucleotide sequence are known in the art.Ribozymes can be designed, for example, as described in PCT Publication Nos. WO93 / 23569 and WO94 / 02595, and can be synthesized and tested in vitro and / or in vivo as described therein.

[0146] Ribozyme activity can be optimized by varying the length of the ribozyme binding arms or chemically synthesizing ribozymes with modifications that prevent degradation by serum RNAses (see, e.g., PCT Publication Nos. WO92 / 07065, WO93 / 15187, WO91 / 03162, and WO94 / 13688; EP92110298.4; and U.S. Pat. No. 5,334,711, which describe various chemical modifications that can be made to the sugar portion of enzymatic RNA molecules), modifications that enhance their effectiveness in cells, as well as removal of stem II bases to shorten RNA synthesis time and reduce chemical requirements.

[0147] G. Immunostimulatory Oligonucleotides In some embodiments, the nucleic acid component of the lipid particles described herein is an immunostimulatory oligonucleotide (ISS; single-stranded or double-stranded) that can induce immune response when administered to a subject, which can be a mammal such as a human.ISS includes, for example, a certain palindrome that results in a hairpin secondary structure (see Yamamoto et al., J. Immunol., 148:4072-6 (1992)) or CpG motif and other known ISS features (e.g., multiple G domains; see PCT Publication No. WO96 / 11266).

[0148] Immunostimulatory nucleic acids are considered to be non-sequence specific if they are not required to specifically bind to and reduce the expression of a target sequence in order to elicit an immune response.Thus, certain immunostimulatory nucleic acids may contain sequences that correspond to a region of a natural gene or mRNA, but still be considered to be non-sequence specific immunostimulatory nucleic acids.

[0149] In one embodiment, the immunostimulatory nucleic acid or oligonucleotide comprises at least one CpG dinucleotide. The oligonucleotide or CpG dinucleotide may be unmethylated or methylated. In another embodiment, the immunostimulatory nucleic acid comprises at least one CpG dinucleotide with a methylated cytosine. In one embodiment, the nucleic acid comprises a single CpG dinucleotide, and the cytosine in the CpG dinucleotide is methylated. In an alternative embodiment, the nucleic acid comprises at least two CpG dinucleotides, and at least one cytosine in the CpG dinucleotide is methylated. In a further embodiment, each cytosine in the CpG dinucleotide present in the sequence is methylated. In another embodiment, the nucleic acid comprises a plurality of CpG dinucleotides, and at least one of the CpG dinucleotides comprises a methylated cytosine. In certain embodiments, the oligonucleotide used in the compositions and methods described herein has a phosphodiester ("PO") or phosphorothioate ("PS") backbone and / or at least one methylated cytosine residue in the CpG motif.

[0150] VI. Preparation of lipid particles The lipid particles described herein, which are capable of encapsulating nucleic acids, such as mRNA, and protecting them from degradation, can be formed by any method known in the art, including, but not limited to, continuous mixing methods and direct dilution processes.

[0151] In some embodiments, the lipid particles described herein are produced through a continuous mixing method, for example, a process that includes preparing an aqueous solution containing nucleic acid in a first reservoir, preparing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution so that the organic lipid solution mixes with the aqueous solution to produce lipid particles that encapsulate nucleic acid substantially instantly.This process and the device for carrying out this process are described in US Patent Publication No. 20040142025.

[0152] The act of continuously introducing lipid and buffer solution into a mixing environment, for example into a mixing chamber, causes the continuous dilution of lipid solution with buffer solution, thereby generating lipid particles substantially immediately when mixed.As used herein, the phrase "continuously dilute lipid solution with buffer solution" (and variations) generally means that lipid solution is diluted sufficiently rapidly in the hydration process with sufficient force to achieve vesicle generation.By mixing the aqueous solution containing nucleic acid with the organic lipid solution, the organic lipid solution undergoes continuous stepwise dilution in the presence of buffer solution (i.e., aqueous solution), generating lipid particles.

[0153] Lipid particles formed using continuous mixing methods typically have a size of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to obtain a uniform particle size.

[0154] In some embodiments, lipid particles as described herein are produced through a direct dilution process, which comprises forming a lipid particle solution and immediately directly introducing the lipid particle solution into a collection vessel that contains a controlled amount of dilution buffer.In certain embodiments, the collection vessel comprises one or more elements that are configured to stir the contents of the collection vessel to facilitate dilution.In one embodiment, the amount of dilution buffer present in the collection vessel is substantially equal to the volume of the lipid particle solution that is introduced therein.As a non-limiting example, it is believed that a 45% ethanol solution of lipid particles advantageously produces smaller particles when introduced into a collection vessel that contains an equal volume of dilution buffer.

[0155] In some embodiments, the lipid particles described herein are produced via a direct dilution process in which a third reservoir containing a dilution buffer is fluidly coupled to the second mixing region. In this embodiment, the lipid particle solution formed in the first mixing region is immediately mixed directly with the dilution buffer in the second mixing region. In certain embodiments, the second mixing region includes a T-connector arranged such that the flows of the lipid particle solution and the dilution buffer meet as 180° opposing flows; however, connectors providing shallower angles, for example, from about 27° to about 180°, may be used. A pump mechanism delivers a controllable flow of buffer to the second mixing region. In one embodiment, the flow rate of the dilution buffer provided to the second mixing region is controlled to be substantially equal to the flow rate of the lipid particle solution introduced thereto from the first mixing region. This embodiment advantageously allows for greater control over the flow of the dilution buffer that mixes with the lipid particle solution in the second mixing region throughout the second mixing process, and therefore also the concentration of the lipid particle solution in the buffer. Such control of the dilution buffer flow rate advantageously allows for the formation of small particle sizes at reduced concentrations.

[0156] These processes and equipment for carrying out these direct dilution processes are described in US Patent Application Publication No. 20070042031.

[0157] Lipid particles formed using the direct dilution process typically have a size of about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, or about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to obtain a uniform particle size.

[0158] If desired, the lipid particles described herein can be sized by any of the methods available to one of skill in the art to achieve a desired size range and relatively narrow particle size distribution.

[0159] Several techniques are available for sizing particles to the desired size. One sizing method used for liposomes and equally applicable to the present particles is described in U.S. Pat. No. 4,737,323. Sonication of a particle suspension, either by bath or probe sonication, results in a gradual reduction in size down to particles less than about 50 nm in size. Homogenization is another method that relies on shear forces to fragment larger particles into smaller ones. In a typical homogenization procedure, particles are recirculated through a standard emulsion homogenizer until a selected particle size is observed, typically between about 60 and about 80 nm. In both methods, the particle size distribution can be monitored by conventional laser beam particle size discrimination or QELS.

[0160] Extrusion of particles through small-pore polycarbonate membranes or asymmetric ceramic membranes is also an effective method for reducing particle size to a relatively well-defined size distribution. Typically, the suspension is cycled through the membrane one or more times until the desired particle size distribution is achieved. Particles can be extruded through successively smaller pore membranes to achieve a stepwise reduction in size.

[0161] In some embodiments, the nucleic acid to lipid ratio (mass / mass ratio) of the formed lipid particles is in the range of about 0.01 to about 0.2, about 0.02 to about 0.1, about 0.03 to about 0.1, or about 0.01 to about 0.08. The ratio of the starting materials also falls within this range. In other embodiments, the preparation of the lipid particles uses a nucleic acid to lipid mass ratio of about 0.04, which corresponds to about 400 μg of nucleic acid per 10 mg of total lipid, or about 0.01 to about 0.08, e.g., 1.25 mg of total lipid per 50 μg of nucleic acid. In certain embodiments, the particles have a nucleic acid:lipid mass ratio of about 0.08.

[0162] In other embodiments, the ratio of lipid to nucleic acid (mass / mass ratio) in the formed lipid particles is from about 1 (1:1) to about 100 (100:1), from about 5 (5:1) to about 100 (100:1), from about 1 (1:1) to about 50 (50:1), from about 2 (2:1) to about 50 (50:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1:1) to about 25 (25:1), from about 2 (2:1) to about 25 (25:1), from about 3 (3:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 5 (5:1) to about 50 (50:1), from about 1 (1 ...4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 4 (4:1) to about 50 (50:1), from about 3:1) to about 25 (25:1), about 4 (4:1) to about 25 (25:1), about 5 (5:1) to about 25 (25:1), about 5 (5:1) to about 20 (20:1), about 5 (5:1) to about 15 (15:1), about 5 (5:1) to about 10 (10:1), about 5 (5:1), 6 (6:1), 7 (7:1), 8 (8:1), 9 (9:1), 10 (10:1), 11 (11:1), 12 (12:1), 13 (13:1), 14 (14:1), or 15 (15:1). The ratio of the starting materials also falls within this range.

[0163] VII. Administration of Lipid Particles Once formed, lipid particles as described herein are useful for introducing nucleic acid such as mRNA into cells.Therefore, the present disclosure also provides a method for introducing nucleic acid such as mRNA into cells.The method is carried out in vitro or in vivo by first forming particles, and then contacting the particles with cells for a sufficient period of time for the delivery of nucleic acid to cells to occur.

[0164] The lipid particles described herein can be adsorbed to almost any cell type that it is mixed with or contacted with.Once adsorbed, the particles can either be endocytosed by a portion of the cells, exchange lipids with the cell membrane, or fuse with the cells.Transfer or incorporation of the nucleic acid portion of the particles can occur via any one of these routes.In particular, when fusion occurs, the membrane of the particle is incorporated into the cell membrane, and the contents of the particle are mixed with intracellular fluid.

[0165] The lipid particles described herein can be administered alone or in admixture with a pharma- ceutically acceptable carrier (e.g., saline or phosphate buffer) selected according to the route of administration and standard pharmaceutical practice. In general, normal buffered saline (e.g., 135-150 mM NaCl) is used as the pharma- ceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.4% saline, 0.3% glycine, etc., including glycoproteins, e.g., albumin, lipoproteins, globulins, etc., for enhanced stability. Further suitable carriers are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coating agents, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, etc. The phrase "pharmaceutical acceptable" refers to molecular entities and compositions that do not produce allergic or similar adverse reactions when administered to humans.

[0166] The pharma- ceutically acceptable carrier is generally added after the particles are formed, thus allowing the particles to be diluted in a pharma- ceutically acceptable carrier, such as normal buffered saline, after they are formed.

[0167] The concentration of particles in a pharmaceutical formulation can vary widely, for example, from less than about 0.05% by weight, usually from about 2 to about 5% by weight or at least about 2 to about 5% by weight, to as much as about 10 to about 90% by weight, and can be selected primarily by fluid volume, viscosity, etc., according to the particular mode of administration selected. For example, the concentration can be increased to reduce the fluid load associated with the procedure. This may be particularly desirable in patients with atherosclerosis-associated congestive heart failure or severe hypertension. Alternatively, particles composed of irritating lipids can be diluted to low concentrations to reduce inflammation at the site of administration.

[0168] The pharmaceutical compositions described herein can be sterilized by conventional well-known sterilization techniques. Aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, and the lyophilized preparation is mixed with a sterile aqueous solution before administration. The composition can contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. In addition, the particle suspension can contain lipid protective agents that protect lipids from free radical and lipid peroxidation damage during storage. Lipophilic free radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators such as ferrioxamine are suitable.

[0169] A. In Vivo Administration In some aspects, the lipid particles described herein are administered to a subject, for example, by systemic delivery to distant target cells via a body system such as the circulation. In certain aspects, the present disclosure provides fully encapsulated lipid particles that protect nucleic acids from nuclease degradation in serum, are non-immunogenic, small in size, and suitable for repeated dosing.

[0170] For in vivo administration, administration may be by any method known in the art, for example, by injection, oral administration, inhalation (e.g., intranasal or intratracheal), transdermal application, or rectal administration. Administration can be accomplished by single or divided doses. The pharmaceutical composition can be administered parenterally, i.e., intraarticularly, intravenously, intraperitoneally, subcutaneously, or intramuscularly. In some embodiments, the pharmaceutical composition is administered intravenously or intraperitoneally by bolus injection (see, e.g., U.S. Pat. No. 5,286,634). Intracellular nucleic acid delivery is also discussed in Straubringer et al., Methods Enzymol., 101:512 (1983); Mannino et al., Biotechniques, 6:682 (1988); Nicolau et al., Crit. Rev. Ther. Drug Carrier Syst., 6:239 (1989); and Behr, Acc. Chem. Res., 26:274 (1993). Still other methods of administering lipid-based therapeutic agents are described, for example, in U.S. Patent Nos. 3,993,754; 4,145,410; 4,235,871; 4,224,179; 4,522,803; and 4,588,578. The lipid particles can be administered by direct injection at the site of disease or by injection at a site distal to the site of disease (see, e.g., Culver, HUMAN GENE THERAPY, MaryAnn Liebert, Inc., Publishers, New York. pp. 70-71 (1994)).

[0171] The compositions described herein, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., can be "nebulized") to be administered via inhalation (e.g., intranasally or intratracheally) (see Brigham et al., Am. J. Sci., 298:278 (1989)). The aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

[0172] In certain embodiments, pharmaceutical compositions can be delivered by intranasal spray, inhalation, and / or other aerosol delivery vehicles.Methods for directly delivering nucleic acid compositions to the lungs via nasal aerosol spray are described, for example, in U.S. Patent Nos. 5,756,353 and 5,804,212.Similarly, drug delivery using intranasal microparticle resins and lysophosphatidyl-glycerol compounds (U.S. Patent No. 5,725,871) is also well known in the pharmaceutical field.Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is ​​described in U.S. Patent No. 5,780,045.

[0173] Formulations suitable for parenteral administration, such as by intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizing agents, thickening agents, stabilizing agents, and preservatives. In certain embodiments, the compositions are administered intravenously (e.g., by intravenous infusion), intramuscularly, pulmonary, orally, topically, intranasally, intracerebrally, intraperitoneally, intravesically, or intrathecally.

[0174] Generally, when administered intravenously, the lipid particle formulation is formulated with a suitable pharmaceutical carrier. Many pharma- ceutically acceptable carriers can be used in the compositions and methods described herein. Formulations suitable for use can be found, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). A variety of aqueous carriers can be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, etc., and the aqueous carrier can include glycoproteins, such as albumin, lipoproteins, globulins, etc., for enhanced stability. Generally, normal buffered saline (135-150 mM NaCl) is used as a pharma- ceutically acceptable carrier, although other suitable carriers will suffice. These compositions can be sterilized by conventional liposomal sterilization techniques, for example, filtration. The compositions may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, humectants, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. These compositions may be sterilized using the techniques mentioned above, but may alternatively be produced under aseptic conditions. The resulting aqueous solutions may be packaged for use or may be filtered and lyophilized under aseptic conditions, and the lyophilized preparation may be combined with a sterile aqueous solution prior to administration.

[0175] In certain applications, the lipid particles described herein can be delivered to a subject by oral administration.The particles can be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, pills, lozenges, elixirs, mouthwashes, suspensions, mouth sprays, syrups, wafers, etc. (see, for example, U.S. Patent Nos. 5,641,515, 5,580,579, and 5,792,451).These oral dosage forms can also include binders, gelatin; excipients, lubricants, and / or flavorings.When the unit dosage form is a capsule, it can include liquid carriers in addition to the above materials.Various other materials can be present as coatings or to otherwise modify the physical form of the dosage unit.Of course, any material used in the preparation of any unit dosage form should be pharma- ceutically pure and substantially non-toxic in the amounts used.

[0176] Typically, these oral formulations can contain at least about 0.1% lipid particles or more, although of course the percentage of particles can vary and can conveniently be between about 1% or about 2% to about 60% or about 70% or more by weight or volume of the total formulation. Of course, the amount of particles in each therapeutically useful composition can be prepared in such a way that an appropriate dosage is obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and therefore various dosages and therapeutic regimens may be desirable.

[0177] Formulations suitable for oral administration may consist of: (a) a liquid solution, such as an effective amount of lipid particles containing nucleic acid (e.g., mRNA) suspended in a diluent such as water, saline, or PEG400; (b) a capsule, sachet, or tablet, each containing a predetermined amount of lipid particles containing nucleic acid (e.g., mRNA) as a liquid, solid, granule, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may contain one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, wetting agents, preservatives, flavoring agents, dyes, disintegrants, and pharma- ceutically compatible carriers. Lozenge forms can include lipid particles containing the nucleic acid (e.g., mRNA) in a flavoring, e.g., sucrose; similarly, pastilles include lipid particles in an inert base, e.g., gelatin and glycerin or sucrose and gum arabic emulsion, gels, and the like, which in addition to the lipid particles include carriers known in the art.

[0178] In another example of their use, the lipid particles can be incorporated into a wide variety of topical dosage forms. For example, suspensions containing the lipid particles can be formulated and administered as gels, oils, emulsions, topical creams, pastes, ointments, lotions, foams, mousses, and the like.

[0179] When preparing pharmaceutical preparations of the lipid particles described herein, it is preferable to use purified, abundant particles to reduce or eliminate empty particles or particles with nucleic acid bound to their outer surface.

[0180] The methods described herein can be carried out in a variety of hosts.Preferred hosts include mammalian species, such as primates (e.g., humans and chimpanzees, as well as other non-human primates), canines, felines, equines, bovines, ovines, caprines, rodents (e.g., rats and mice), lagomorphs, and porcines.

[0181] The amount of particles administered will depend on the ratio of nucleic acid (e.g., mRNA) to lipid, the particular nucleic acid used, the disease or disorder being treated, the age, weight, and condition of the subject, and the judgment of the clinician, but will generally be between about 0.01 mg / kg and about 50 mg / kg body weight, between about 0.1 mg / kg and about 5 mg / kg body weight, or about 10 mg / kg per administration (e.g., injection). 8 ~10 10 It is thought to be a particle.

[0182] B. In Vitro Administration For in vitro applications, delivery of nucleic acid (e.g., mRNA) can be to any cell grown in culture, whether of plant or animal origin, vertebrate or invertebrate, of any tissue or type. In certain embodiments, the cell is an animal cell, e.g., a mammalian cell, e.g., a human cell.

[0183] Contact between cells and lipid particles, when performed in vitro, generally occurs in a biologically compatible medium. The concentration of particles can vary widely depending on the particular application, but is generally between about 1 μmol and about 10 mmol. Treatment of cells with lipid particles is generally performed at physiological temperature (about 37° C.) for a period ranging from about 1 to about 48 hours, for example, from about 2 to about 4 hours.

[0184] In some embodiments, the lipid particle suspension comprises about 10 3 ~about 10 5 Cells / ml, e.g., about 2 x 10 4 The suspension is added to 60-80% confluent plated cells having a cell density of about 10 ...

[0185] Endosomal release parameter (ERP) assays can be used to optimize lipid particle delivery efficiency. ERP assays are described in US Patent Publication No. 20030077829. More specifically, the purpose of the ERP assay is to distinguish the effects of various cationic lipid and helper lipid components based on their relative impact on endosomal membrane binding / uptake or fusion with / destabilization of endosomal membrane. This assay allows for quantitative determination of how each component of the lipid particle affects delivery efficiency, thereby optimizing the lipid particle. Typically, ERP assays measure the expression of a reporter protein (e.g., luciferase, beta-galactosidase, green fluorescent protein (GFP), etc.), and in some cases, lipid particle formulations optimized for expression plasmids may also be suitable for encapsulating other types of nucleic acids, such as mRNA. In some cases, ERP assays can be adapted to measure downregulation of transcription or translation of a target sequence in the presence or absence of an interfering RNA (e.g., siRNA). In other examples, ERP assays can be adapted to measure expression of a target protein in the presence or absence of mRNA. By comparing the ERP for each of a variety of lipid particles, an optimized system, for example lipid particles with maximum uptake into cells, can be readily determined.

[0186] C. Cells for Delivery of Lipid Particles The compositions and methods described herein are used to treat a wide variety of cell types in vivo and in vitro. Suitable cells include, for example, hematopoietic progenitor (stem) cells, fibroblasts, keratinocytes, hepatocytes, immune cells, endothelial cells, skeletal and smooth muscle cells, osteoblasts, neurons, resting lymphocytes, terminally differentiated cells, slow or noncycling primary cells, parenchymal cells, lymphoid cells, epithelial cells, bone cells, etc. In some embodiments, lipid particles containing nucleic acids (e.g., mRNA) are delivered to immune cells, such as, for example, antigen-presenting cells (e.g., dendritic cells, macrophages, B cells) and T cells (e.g., helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells). In some embodiments, lipid particles comprising a nucleic acid (e.g., siRNA) are delivered to cancer cells, such as, for example, lung cancer cells, colon cancer cells, rectal cancer cells, anal cancer cells, cholangiocarcinoma cells, small intestine cancer cells, gastric cancer cells, esophageal cancer cells, gallbladder cancer cells, liver cancer cells, pancreatic cancer cells, appendix cancer cells, breast cancer cells, ovarian cancer cells, cervical cancer cells, prostate cancer cells, renal cancer cells, cancer cells of the central nervous system, glioblastoma tumor cells, skin cancer cells, lymphoma cells, choriocarcinoma tumor cells, head and neck cancer cells, osteogenic sarcoma tumor cells, and blood cancer cells.

[0187] The in vivo delivery of lipid particles containing nucleic acid is suitable for targeting any cell type of cells.The method and composition can be used in a wide variety of vertebrate cells, including mammals, such as canine, feline, equine, bovine, ovine, caprine, rodent (e.g., mouse, rat, and guinea pig), lagomorph, porcine, and primate (e.g., monkey, chimpanzee, and human). EXAMPLES

[0188] The present disclosure will now be described in more detail by way of examples. The following examples are provided for illustrative purposes only and are not intended to limit the present disclosure in any manner.

[0189] Example 1. Synthesis of ditetradecylamine for poly(alkyloxazoline)-lipid conjugates Ditetradecylamine (5) was synthesized according to the scheme shown below. TIFF2025504682000018.tif31129

[0190] Synthesis of 4-methyl-N,N-ditetradecylbenzenesulfonamide (3). K2CO3 (20.18 g, 146.0 mmol), p-toluenesulfonamide (1) (5.00 g, 29.2 mmol) and 1-bromotetradecane (2) (21.7 ml, 73.0 mmol) were heated to reflux in DMF (75 ml) for 20 h. The reaction was cooled to room temperature, diluted with Et2O (100 ml) and filtered. The filtrate was washed with H2O (50 ml) and the aqueous solution was back-extracted with Et2O (100 ml). The combined organics were dried (Na2SO4), filtered and concentrated in vacuo and the residue was purified by automated flash chromatography (DCM / hexane 40 / 60) to give 4-methyl-N,N-ditetradecyl-p-toluenesulfonamide (3) (15.86 g, 96%).

[0191] Synthesis of ditetradecylamine (5). A solution of 4-methyl-N,N-ditetradecylbenzenesulfonamide (3) (10 g, 17.7 mmol) in THF (5 mL) was added to a solution of lithium naphthalenide (prepared by stirring naphthalene (4) (11.36 g, 88.7 mmol) and lithium metal (0.92 g, 133.0 mmol) in THF (40 mL) at room temperature for 1 h) at 0 °C, and the reaction was stirred at room temperature for 1 h. MeOH (4 mL) was added, followed by H2O (50 mL), and the mixture was extracted with ether (100 mL). The organics were dried (Na2SO4), filtered, and concentrated in vacuo. The residue was purified by automated flash chromatography (EtOAc / Hex (50:50)) to give ditetradecylamine (5) (4.85 g, 66.7%).

[0192] Example 2. Synthesis of 2,3-bis(tetradecyloxy)propan-1-amine for poly(alkyloxazoline)-lipid conjugates 2,3-Bis(tetradecyloxy)propan-1-amine (TFA salt) (10) was synthesized according to the following scheme. TIFF2025504682000019.tif26159

[0193] Synthesis of tert-butyl (2,3-dihydroxypropyl)carbamate (8). Di-t-butyl dicarbonate (7) (14.23 g, 65.2 mmol) and 3-amino-1,2-propanediol (6) (5.4 g, 59.3 mmol) were stirred in DCM / MeOH (1:1) at room temperature for 16 h. The reaction was concentrated in vacuo to give 8 (11.25 g, 99%) which was used without purification.

[0194] Synthesis of tert-butyl(2,3-bis(tetradecyloxy)propyl)carbamate (9). tert-Butyl(2,3-dihydroxypropyl)carbamate (8) (5 g, 26.2 mmol), bromotetradecane (21.75 g, 78.5 mmol), and TBAHS (4.44 g, 13.1 mmol) were stirred in toluene (20 ml) at 0° C. NaOH (15 mL, 50% w / v) was added and the biphasic mixture was stirred vigorously for 18 h, warming to room temperature. The reaction was diluted with water and extracted with hexanes. The combined organics were washed (brine), dried (MgSO4), filtered, and concentrated in vacuo. The crude material was chromatographed (10% EtOAc-hexanes) to give tert-butyl(2,3-bis(tetradecyloxy)propyl)carbamate (9) (quantitative).

[0195] Synthesis of 2,3-bis(tetradecyloxy)propan-1-amine (TFA salt) (10). tert-Butyl (2,3-bis(tetradecyloxy)propyl)carbamate (9) prepared in step 2 was stirred in TFA / DCM (1:1) at room temperature for 2 h. The reaction was concentrated in vacuo to give 2,3-bis(tetradecyloxy)propan-1-amine (TFA salt) (10) (8.3 g, quantitative), which was used without purification.

[0196] Example 3. Synthesis of poly(alkyloxazoline)-lipid conjugates For conjugate (11), an N-alkylated thiopropanamide POZ lipid was synthesized as shown below. TIFF2025504682000020.tif23164

[0197] The reagents were stirred in DCM at room temperature for 16 h. The reaction was concentrated in vacuo and the residue was purified by automated flash chromatography (0-25% MeOH / DCM). Similar product containing fractions were combined and concentrated in vacuo. The residue was dissolved in a minimum amount of DCM and Et2O was added to give a precipitate which was collected by filtration to give 11 (38 mg, 11.7%). TIFF2025504682000021.tif12159

[0198] The following lipid conjugates were prepared using similar methodology as above with the appropriate amines. TIFF2025504682000022.tif190159

[0199] Example 4. General method for preparation and characterization of lipid nanoparticles Lipid Nanoparticle (LNP) Formulations The lipid solution contains four components: polymer-conjugated lipid, ionizable cationic lipid (e.g., (6Z,16Z)-12-((Z)-dec-4-en-1-yl)docosa-6,16-dien-11-yl 5-(dimethylamino)-pentanoate), cholesterol, and phospholipid (e.g., DSPC). Lipid stocks were prepared using the lipid identities and molar ratios listed to a total concentration of approximately 7 mg / mL in 100% ethanol. Firefly luciferase (Luc) mRNA (TriLink Biotechnologies, L-7202) or ovalbumin (OVA) mRNA (TriLink Biotechnologies, L-7210) was diluted in pH 5 acetate buffer and nuclease-free water to a target concentration of 0.366 mg / mL mRNA in 100 mM acetic acid, pH 5. Equal volumes of lipid and nucleic acid solutions were mixed through a T-connector at a flow rate of 400 mL / min and diluted with approximately 4 volumes of PBS, pH 7.4. The formulation was placed in a Slide-A-Lyzer dialysis unit (MWCO 10,000) and dialyzed overnight against 10 mM Tris, 500 mM NaCl, pH 8 buffer. Following dialysis, the formulation was concentrated to approximately 0.5 mg / mL using a VivaSpin concentrator unit (MWCO 100,000) and dialyzed overnight against 5 mM Tris, 10% sucrose, pH 8 buffer. The formulation was filtered through a 0.2 μm syringe filter and stored at −80° C. until use. Nucleic acid concentration was determined by RiboGreen assay. Particle size (hydrodynamic diameter) and polydispersity index (PDI) were determined by dynamic light scattering measurements using a Malvern Nano Series Zetasizer.

[0200] In vivo testing of lipid nanoparticles via intravenous administration (luciferase model) LNP formulations encapsulating firefly luciferase were injected intravenously at 0.5 mg / kg into female BALB / c mice (6-8 weeks old). On the day of injection, LNP stocks were filtered and diluted to the required dosing concentration in phosphate buffered saline, pH 7.4. Six hours after dose administration, animals were anesthetized with a lethal dose of ketamine / xylazine. Blood samples were collected in EDTA microtiter tubes and centrifuged at 16,000xg for 5 min at 4°C. All plasma samples were stored at -80°C until cytokine analysis. Liver samples were collected, weighed, and flash frozen in liquid nitrogen. Liver samples were stored in FastPrep® tubes at -80°C until analysis of luciferase activity.

[0201] Cytokine analysis Proinflammatory cytokine (i.e., monocyte chemotactic protein-1, MCP-1) production was measured by ELISA using pair-matched monoclonal antibodies from BD Biosciences. Plates were coated with capture antibody and stored overnight at 2-8 °C. The next day, plates were blocked with 10% FBS in 1x PBS and test samples were added. After three washes, detection antibody was added and incubated for 1 h. After incubation and washing, TMB substrate was added to develop color and the reaction was stopped with sulfuric acid. Plates were read at OD 450 / 570.

[0202] Luciferase activity analysis Frozen aliquots of liver were thawed and homogenized in 1 mL of 1×CCLR (Cell Culture Lysis Reagent) using a FastPrep® homogenizer. The homogenate was centrifuged at 16,000 rpm for 10 min at 4° C. 20 μL of the supernatant was placed in a 96-well white plate and luminescence was measured following the addition of luciferase reagent (Promega Luciferase Assay System). Luciferase activity was determined by comparing the luminescence of homogenized samples to a luciferase protein standard. To account for any quenching of luminescence by components in the liver homogenate, luciferase was added to liver homogenates of untreated animals and the resulting luminescence was measured. A quenching factor was applied to all samples to obtain corrected luciferase activity values, which were normalized per unit mass of tissue analyzed.

[0203] In vivo testing of lipid nanoparticles via intramuscular administration (ovalbumin immunogenicity model) LNP formulations encapsulating OVA mRNA were administered intramuscularly at 1 μg doses to BALB / c mice (7-8 weeks old) on days 0 (prime) and 21 (boost). Blood samples were collected in EDTA microtiter tubes prior to administration and on days 7, 14, 21, and 28. On day 35, animals were anesthetized with a lethal dose of ketamine / xylazine. Blood samples were collected in EDTA microtiter tubes and centrifuged at 16,000 x g for 5 min at 4 °C. All plasma samples were stored at -80 °C until anti-OVA IgG ELISA was performed. Spleen samples were also collected at the final time point, and splenocytes were harvested for ELISpot assays.

[0204] Anti-OVA IgG analysis The production of anti-OVA IgG antibodies was measured using an ELISA method. Plates were coated with ovalbumin protein and stored overnight at 2-8°C. The next day, plates were blocked with 10% FBS in PBS and samples were added. After washing, secondary antibodies were added and incubated for 2 hours at room temperature. Following incubation, plates were washed and TMB substrate was added to develop the color. The reaction was stopped with 2N sulfuric acid and plates were read at OD 450 / 570.

[0205] ELISpot analysis At the end of the time point, spleen samples were collected from the animals and splenocytes were isolated. ELISpot plates were prepared by washing the plates and incubating with medium for at least 30 minutes. Following incubation, medium was removed and stimuli were added. Cells were added and incubated overnight at 37°C. After overnight incubation, cells were removed and plates were washed. Detection antibody was added followed by incubation at room temperature for 2 hours. Plates were washed and streptavidin-HRP antibody was added. After incubation at room temperature for 1 hour, plates were washed and TMB substrate was added to develop color. The reaction was stopped with 2N sulfuric acid and plates were read at OD 450 / 570.

[0206] Example 5. Particle characteristics of LNP formulations containing poly(2-ethyl 2-oxazoline) (PEOZ)-lipid conjugates PEOZ lipids of approximately 5000 Da with different alkyl chain lengths and linkers were formulated into LNPs and compared with two PEG-lipid controls. Luc mRNA was used as the payload. Despite the hydrophobic nature of the PEOZ 5000 polymer, its incorporation into LNPs resulted in the formation of stable particles. Importantly, LNPs formulated with different chemistries of PEOZ lipids reported physicochemical properties comparable to those prepared with PEG2000-C-DMA and commercially available PEG-DMG lipids (Table 1).

[0207] Table 1. Particle characteristics of LNP formulations containing various PEOZ lipids. TIFF2025504682000023.tif54166%EE=encapsulation efficiency;PDI=polydispersity index

[0208] Example 6. Performance of LNPs containing PEOZ-lipid conjugates in an intravenous mouse model LNP formulations carrying luciferase mRNA payload and PEOZ5000 lipid were compared to reference controls (PEG2000-C-DMA and PEG-DMG) for activity in a luciferase mouse model after intravenous administration. Six hours after administration, PEOZ lipid formulations mediated high levels of luciferase activity in the liver, with comparable or improved activity reported compared to controls (Table 2). Furthermore, these PEOZ lipid formulations reported lower luciferase expression in the spleen compared to commercial PEG-DMG, indicating lower non-specific (off-target) activity (Table 3).

[0209] Table 2. Luciferase activity in liver 6 hours after intravenous administration of 0.5 mg / kg LNPs containing PEG- or PEOZ-conjugated lipids in BALB / c mice (n=4). TIFF2025504682000024.tif62166

[0210] Table 3. Luciferase activity in the spleen 6 hours after intravenous administration of 0.5 mg / kg LNPs containing PEG- or PEOZ-conjugated lipids in BALB / c mice (n=4). TIFF2025504682000025.tif62166

[0211] To assess the immunostimulatory potential of these PEOZ formulations, MCP-1 induction was measured in plasma samples 6 hours after intravenous administration. PEOZ-based LNPs induced similar or lower cytokine levels compared to their established PEGylated counterparts (Table 4).

[0212] Table 4. MCP-1 induction 6 hours after intravenous administration of 0.5 mg / kg luciferase mRNA-LNPs containing PEG- or PEOZ-conjugated lipids in BALB / c mice (n=4). TIFF2025504682000026.tif68166

[0213] Example 7. Effect of PEOZ-lipid on the clearance rate in plasma LNP formulations carrying approximately 5000 Da PEOZ lipid were compared to a reference control (PEG2000-C-DMA) for clearance rate from plasma after intravenous injection. Mass spectrometry was used to assess the rate of clearance using the ionizable lipid level in the composition as a surrogate. At an LNP ratio of polymer conjugate lipid:cationic lipid:cholesterol:DPSC of 1.6:54.6:32.8:10.9, PEOZ-containing formulations (compounds 11, 12, 13, 14, and 15) showed enhanced clearance rate from plasma at 0.25 hours after injection compared to the reference (Table 5). The shorter residence time in the plasma compartment exhibited by PEOZ-containing LNPs could potentially help mitigate antibody responses upon repeated dose administration.

[0214] Table 5: Plasma clearance rate after intravenous injection of 0.5 mg / kg luciferase mRNA-LNPs containing PEG- or PEOZ-conjugated lipids in BALB / c mice (n=4). Blood samples were collected at 15 min, 30 min, 1 h, 3 h, and 6 h after administration. TIFF2025504682000027.tif65166

[0215] Example 8. Effect of PEOZ-lipid ratio and structure on particle properties PEOZ lipids of about 5000 Da with dialkyl C14 chains were formulated into LNPs at various molar ratios (0.5%, 1.5%, and 3.0%). Despite the hydrophobicity of the PEOZ5000 polymer, its incorporation into LNPs resulted in the formation of stable particles, the size of which was dependent on the amount of PEOZ lipid. As the amount of PEOZ lipid increased, the particle size decreased (Table 6). At a fixed PEOZ lipid concentration of 1.5 mol%, varying the chemical nature of the PEOZ lipid did not significantly affect the particle properties (Table 7). Taken together, these results demonstrate that PEOZ formulations can be engineered by varying the chain length and molar ratio.

[0216] Table 6. Particle characteristics of ovalbumin mRNA-LNP formulations containing titrations of PEOZ5000-DMA lipids TIFF2025504682000028.tif36165%EE=encapsulation efficiency;PDI=polydispersity index

[0217] Table 7. Particle characteristics of ovalbumin mRNA-LNP formulations containing PEOZ lipids TIFF2025504682000029.tif50166%EE=encapsulation efficiency;PDI=polydispersity index

[0218] Example 9. Effect of lipid ratio on immunogenicity of PEOZ-LNPs in the ovalbumin model LNP formulations containing various amounts of PEOZ5000 lipid were evaluated for their ability to induce antigen-specific immunogenicity following intramuscular (IM) administration in an ovalbumin (OVA) model. This model defines the efficacy of the formulations in a vaccine setting. Formulations were compared to a PEG-lipid reference control.

[0219] Animals were treated with LNPs in the prime / boost regimen described in Example 4 and then evaluated for the level of anti-OVA IgG antibodies produced on day 35. LNPs containing 0.5% PEOZ produced comparable levels of anti-OVA IgG compared to the reference control (Table 8). These trends were followed through the analysis of interferon gamma-producing splenocytes (Table 9). Alternative PEOZ-lipid structures were also tested and resulted in comparable levels of anti-OVA IgG levels (Table 10), and similar or higher levels of interferon gamma-producing splenocytes (Table 11), compared to the reference.

[0220] These results demonstrate that LNPs containing PEOZ-lipids can be used to deliver mRNA encoding antigens and generate a host immune response against these antigens, and therefore can be successfully incorporated into a vaccine platform. PEG is found in many other products, such as cosmetics, and has reportedly caused some populations to produce antibodies against PEG. This can cause adverse reactions when such individuals are exposed to PEG in the form of LNPs, for example, coronavirus vaccines (Shimabukuro 2021). Therefore, it would be highly useful to be able to replace the PEG lipids in LNPs with alternative polymer-conjugated lipids, such as PEOZ-lipids.

[0221] Table 8. Anti-OVA IgG levels at 35 days after intramuscular administration of a 1 μg dose of OVA mRNA LNP on days 0 and 21 in BALB / c mice (n=4). TIFF2025504682000030.tif43166

[0222] Table 9. IFN-gamma producing spleen cells at 35 days after intramuscular administration of a 1 μg dose of OVA mRNA LNP on days 0 and 21 in BALB / c mice (n=4). TIFF2025504682000031.tif43166

[0223] Table 10. Anti-OVA IgG levels at 35 days after intramuscular administration of a 1 μg dose of OVA mRNA LNP on days 0 and 21 in BALB / c mice (n=4). TIFF2025504682000032.tif57165

[0224] Table 11. IFN-gamma producing spleen cells at 35 days after intramuscular administration of a 1 μg dose of OVA mRNA LNP on days 0 and 21 in BALB / c mice (n=4). TIFF2025504682000033.tif57165

[0225] It should be understood that the above description is intended to be illustrative and not limiting. Many aspects will become apparent to those skilled in the art upon reading the above description. Therefore, the scope of the present invention should not be determined with reference to the above description, but should instead be determined with reference to the appended claims, together with the full scope of equivalents to which such claims are entitled. The disclosures of all papers and references, including patent applications, patents, PCT publications, and Genbank accession numbers, are incorporated herein by reference for all purposes.

Claims

1. Poly(alkyloxazoline)-lipid conjugates according to formula I: or a pharma- ceutically acceptable salt thereof, During the ceremony: The subscript n is an integer ranging from 10 to 100; R 1 is independently C 1～6 is alkyl; Each R 2 is hydrogen, C 1～6 Alkyl, and C 1～6 acyl; (i) Z is -S(CH 2 ) 2 C(O)-, R 3 -NR 3a R 3b , -NR 3a CH 2 CH(R 3b ) 2 , -CH(R 3b ) 2 , and -CH 2 CH(R 3b ) 2 selected from the group consisting of: or (ii) Z is -Z 1 -OC(O)-, -Z 1 -NHC(O)-, -Z 1 -S(O) 2 - and -Z 1 -OCH 2 - selected from the group consisting of Z 1 is selected from the group consisting of a covalent bond, an oligo(ethylene glycol) diradical, and a poly(ethylene glycol) diradical; R 3 -NR 3a R 3b and -CH(R 3b ) 2 selected from the group consisting of: In the formula, each R 3a and R 3b is hydrogen, C 6～22 Alkyl, C 6～22 Alkenyl, and C 6～22 alkynyl; R 3a and R 3b One or more non-adjacent CHs in 2 The groups may be independently replaced with oxygen; However, at least one R 3a or R 3b But, C 6～22 Alkyl, C 6～22 Alkenyl, and C 6～22 alkynyl; R 3a or R 3b One or more non-adjacent CHs in 2 groups are optionally independently replaced with oxygen; The poly(alkyloxazoline)-lipid conjugate, or a pharma- ceutically acceptable salt thereof.

2. R 1 The poly(alkyloxazoline)-lipid conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein is ethyl.

3. R 1 The poly(alkyloxazoline)-lipid conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein is methyl.

4. Each R 1 The poly(alkyloxazoline)-lipid conjugate of claim 1, or a pharma- ceutically acceptable salt thereof, wherein is independently selected from the group consisting of ethyl and methyl.

5. The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 4, wherein the subscript n is an integer ranging from about 15 to about 55, or a pharma- ceutically acceptable salt thereof.

6. 6. The poly(alkyloxazoline)-lipid conjugate of claim 5, or a pharma- ceutically acceptable salt thereof, wherein the poly(alkyloxazoline) portion of the conjugate has a number average molecular weight ranging from about 2,000 Da to about 5,000 Da.

7. Formula IIa: The structure is During the ceremony: R 2 is selected from the group consisting of hydrogen and methyl; R 3a is hydrogen, C 12～18 Alkyl, and C 12～18 alkenyl; R 3b is C 12～18 Alkyl and C 12～18 alkenyl, The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof.

8. R 3a and R 3b But, C 12～18 Alkyl and C 12～18 The poly(alkyloxazoline)-lipid conjugate of claim 7, wherein said poly(alkyloxazoline)-lipid conjugate is independently selected from the group consisting of alkenyl.

9. Formula IIb: The structure is During the ceremony: R 2 is selected from the group consisting of hydrogen and methyl; Each R 3c is C 12～18 Alkyl and C 12～18 alkenyl, The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, or a pharma- ceutically acceptable salt thereof.

10. Z is -Z 1 -OC(O)- and Z 1 is selected from the group consisting of a covalent bond and a poly(ethylene glycol) diradical; R 3 Ga-NR 3a R 3b The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

11. Z is -Z 1 -NHC(O)-, and Z 1 is a covalent bond, and R 3 Ga-NR 3a R 3b The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

12. Z and R 3 Ga-Z 1 -OC(O)- and Z 1 is a covalent bond, and R 3 -CH(R 3b ) 2 The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

13. Z is -Z 1 -S(O) 2 - and Z 1 is a covalent bond, and R 3 Ga-NR 3a R 3b The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

14. Z is -Z 1 -OCH 2 - and Z 1 is a covalent bond, and R 3 -CH(R 3b ) 2 The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

15. Z is -Z 1 -NHC(O)-, and Z 1 is a covalent bond, and R 3 -CH(R 3b ) 2 The poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 6, wherein

16. R 3a and each R 3b But, C 12～18 Alkyl and C 12～18 The poly(alkyloxazoline)-lipid conjugate of any one of claims 10 to 15, wherein said poly(alkyloxazoline)-lipid conjugate is independently selected from the group consisting of alkenyl.

17. A lipid nanoparticle comprising the poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 16.

18. 18. The lipid nanoparticle of claim 17, further comprising a cationic lipid, a neutral lipid, a sterol, or a combination thereof.

19. The lipid nanoparticle of claim 17 or 18, further comprising a nucleic acid.

20. A lipid nanoparticle, comprising: (a) Nucleic acid; (b) a cationic lipid constituting 30 mol % to 80 mol % of the total lipid present in the lipid nanoparticle; (c) Neutral lipid; (d) sterols; and (e) a poly(alkyloxazoline)-lipid conjugate conjugate constituting 0.1 mol % to 10 mol % of the total lipid present in the lipid nanoparticle.

21. The lipid nanoparticle of claim 20, wherein the cationic lipid constitutes 40 mol% to 70 mol% of the total lipid present in the lipid nanoparticle.

22. The lipid nanoparticle of claim 20 or 21, wherein the cationic lipid constitutes 45 mol% to 65 mol% of the total lipid present in the lipid nanoparticle.

23. The lipid nanoparticle of any one of claims 20 to 22, wherein the cationic lipid constitutes 45 mol% to 60 mol% of the total lipid present in the lipid nanoparticle.

24. The lipid nanoparticle of any one of claims 20 to 23, wherein the cationic lipid constitutes 50 mol% to 60 mol% of the total lipid present in the lipid nanoparticle.

25. The lipid nanoparticle of any one of claims 20 to 24, wherein the neutral lipid constitutes 3 mol% to 20 mol% of the total lipid present in the lipid nanoparticle.

26. The lipid nanoparticle of any one of claims 20 to 25, wherein the neutral lipid constitutes 5 mol% to 15 mol% of the total lipid present in the lipid nanoparticle.

27. The lipid nanoparticle of any one of claims 20 to 26, wherein the neutral lipid constitutes 8 mol% to 12 mol% of the total lipid present in the lipid nanoparticle.

28. The lipid nanoparticle of any one of claims 20 to 27, wherein the sterol constitutes 10 mol% to 60 mol% of the total lipid present in the lipid nanoparticle.

29. The lipid nanoparticle of any one of claims 20 to 28, wherein the sterol constitutes 20 mol% to 50 mol% of the total lipid present in the lipid nanoparticle.

30. The lipid nanoparticle of any one of claims 20 to 29, wherein the sterol constitutes 30 mol% to 40 mol% of the total lipid present in the lipid nanoparticle.

31. The lipid nanoparticle of any one of claims 20 to 30, wherein the poly(alkyloxazoline)-lipid conjugate constitutes 0.1 mol% to 5 mol% of the total lipid present in the lipid nanoparticle.

32. The lipid nanoparticle of any one of claims 20 to 31, wherein the poly(alkyloxazoline)-lipid conjugate constitutes 0.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticle.

33. The cationic lipid constitutes 50 mol% to 60 mol% of the total lipid present in the lipid nanoparticle, the neutral lipid constitutes 8 mol% to 12 mol% of the total lipid present in the lipid nanoparticle, the sterol constitutes 30 mol% to 40 mol% of the total lipid present in the lipid nanoparticle, and the poly(alkyloxazoline)-lipid conjugate constitutes 0.5 mol% to 3 mol% of the total lipid present in the lipid nanoparticle. The lipid nanoparticle of claim 20.

34. The lipid nanoparticle of any one of claims 20 to 33, wherein the neutral lipid comprises a phospholipid.

35. The lipid nanoparticle of any one of claims 20 to 34, wherein the sterol comprises cholesterol.

36. The lipid nanoparticle of any one of claims 20 to 35, wherein the poly(alkyloxazoline)-lipid conjugate comprises a poly(alkyloxazoline)-lipid conjugate of any one of claims 1 to 16.

37. The lipid nanoparticle of any one of claims 19 to 35, wherein the nucleic acid comprises RNA.

38. The lipid nanoparticle of claim 37, wherein the RNA comprises mRNA.

39. The lipid nanoparticle of any one of claims 19 to 38, wherein the nucleic acid is fully encapsulated in the lipid nanoparticle.

40. The lipid nanoparticle of any one of claims 17 to 39, wherein the lipid nanoparticle has an average diameter in the range of 40 nm to 150 nm.

41. A pharmaceutical composition comprising the lipid nanoparticles of any one of claims 17 to 40 and a pharma- ceutically acceptable carrier.

42. 42. The pharmaceutical composition of claim 41, formulated for intravenous, intramuscular, pulmonary, intracerebral, intrathecal, or intranasal administration.

43. 1. A method for introducing a nucleic acid into a cell, comprising: The method comprising the step of contacting said cells with the lipid nanoparticles of any one of claims 17 to 40 or the pharmaceutical composition of claim 41 or 42.

44. 1. A method for delivering a nucleic acid to a subject, comprising: The method comprising the step of administering to the subject the lipid nanoparticles of any one of claims 17 to 40 or the pharmaceutical composition of claim 41 or 42.

45. 1. A method for preventing or treating a disease or disorder in a subject in need thereof, comprising: The method comprising the step of administering to the subject the lipid nanoparticles of any one of claims 17 to 40 or the pharmaceutical composition of claim 41 or 42.

46. 46. ​​The method of claim 45, wherein the disease or disorder is a viral infection, a liver disease or disorder, a lung disease or disorder, a disease or disorder of the CNS, or cancer.

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